Top 8 Methodologies Neces­sary for Genetic Research

The following points highlight the top eight methodologies necessary for genetic research. The methodologies are:- 1. Isolation of Plasmids 2. Isolation of Chromosomal DNA 3. Agarose Gel Electrophoresis 4. Restriction and Ligation 5. Transformation 6. Polyacrylamide Gel Electrophoresis 7. Two-Dimensional Electrophoresis and 8. Immunoelectrophoresis.

Methodology # 1. Isolation of Plasmids:

Plasmids are self-replicating, extrachro-mosomal genetic elements found in bacteria. They are double-stranded, closed circular (CC) molecules of DNA ranging in size from 1 kb to 200 kb. They usually carry genes which con­fer resistance to antibiotics and for the pro­duction of toxins such as colicin.

In molecular biology, plasmids are particu­larly useful in DNA cloning. Many small plas­mids are maintained at a high copy number within the cell. In contrast to the host chro­mosome, some plasmids can replicate in the absence of protein synthesis. This ability allows plasmid amplification up to a level of several thousand copies per cell by growing plasmid-containing cells in the presence of chloramphenicol.

These prop­erties not only permit the relatively easy isola­tion of plasmid DNA in large amounts, but also allow the synthesis of large quantities of plasmid-encoded products or the products of DNA fragments cloned in them.

To be useful as a cloning vector, a plasmid should be relatively small (to have a high copy number) and should replicate in a relaxed manner. It should carry one or more selectable markers to allow identification of transformants and to maintain the plasmid within the bacterial cell.

Finally, it should contain a single recognition site for one or more restriction enzymes in regions of the plasmid that are not essential for replication. These restriction sites, into which foreign DNA can be inserted, should preferably be located within the genes coding for selectable markers so that insertion of a foreign DNA fragment inactivates the gene.

The most widely used cloning vector is the plasmid, pBR322. This is a plasmid under relaxed control containing ampicillin and tetra­cycline resistance genes (Fig. 1). It has single sites for many restriction enzymes and is small in size, 4.3 kb. The nucleotide sequence of this plasmid is also known.

Large Scale Isolation of Plasmid DNA:

The procedure given below for the isola­tion of plasmid DNA is a modification of the method described by Maniatis (1982).

Plasmids are usually maintained in bacteri­al hosts such as Escherichia coli. The chro­mosomal DNA of the bacterium must there­fore be removed during the preparation of plasmid DNA. Since E. coli chromosome is much larger than plasmid DNA, during the extraction procedure described here, the chro­mosomal DNA is broken down to smaller lin­ear molecules which are then precipitated and removed along with the remnants of lysed cells.

The closed circular molecules of the plas­mid, however, remain in solution. The plasmid DNA is purified further from any contaminat­ing chromosomal DNA by centrifuging to equilibrium in caesium chloride gradients con­taining ethidium bromide. Ethidium bromide is a dye which intercalates between DNA base pairs and in doing so, causes DNA to unwind.

The fragmented chromosomal DNA is capable of binding more ethidium bromide than the closed circular plasmid DNA. This differential binding of the dye to the two species of DNA makes plasmid DNA denser than chromoso­mal DNA, thus enabling plasmid DNA to be isolated in a relatively pure form.

Reagents:

Solution A:

Lysozyme – 12 mg

Glucose (20%, w/v) – 0.276 ml

Na₂EDTA (0.1 M) – 0.60 ml

Tris-HCl, pH 8 (1 M) – 0.15 ml

Distilled water – 4.96 ml

Solution B:

NaOH (2 M) – 6 ml

SDS (10%, w/v) – 6 ml

Distilled water – 48 ml

Solution C:

Sodium acetate (3 M), pH 4.8 – 2.5 ml

tRNA (200 ug/ml) – 0.05 ml

Solution D (TE buffer):

Tris-HCl, pH 8 (1 M) – 0.1 ml

Na₂EDTA (0.1 M) – 0.1 ml

LG Broth

Tryptone – 1 g

Yeast extract – 0.5 g

NaCl – 0.5 g

Glucose – 0.2 g

Distilled water – 100 ml

Autoclaved for 20 minutes at 15 lbs/sq. in and 121°C.

Preparation of plasmid DNA for density gradient centrifugation:

1. Inoculate 20 ml LG broth containing ampicillin (100 µg/ml) with an E. coli cul­ture harboring the plasmid, pBR322, and incubate the culture overnight at 37°C.

2. Using the 20 ml culture as the inoculum, inoculate 2 × 500 ml fresh LG broth con­taining ampicillin (100 ug/ml) and grow the culture at 37°C to an O.D. (600 nm) of 0.8.

3. Add chloramphenicol (75 mg/500 ml cul­ture) and continue incubating the culture overnight with shaking.

4. Harvest the bacterial cells the following day by spinning at 5,000 r.p.m. for 15 minutes at 4°C using 4 × 250 ml bottles.

5. Resuspend the bacterial pellets in 4 x 5 ml of Solution A.

6. Transfer into 4 × 50 ml centrifuge tubes and let the suspensions stand at room temperature for 5 minutes.

7. Add 10 ml of Solution B into each tube mix gently and stand on ice for 10 min­utes.

8. Add 7.5 ml of an ice-cold solution of Solution C to each tube. Mix thoroughly. Stand on ice for 15 minutes.

9. Spin at 18,000 r.p.m. for 30 minutes and 4°C. (The chromosomal DNA and bacte­rial debris should form a tight pellet).

10. Pool the supernatants into fresh 50 ml centrifuge tubes and for every 10 ml of supernatant, add 6 ml iso-propanol. Mix well and allow to stand at room tempera­ture for 30 minutes.

11. Spin at 18,000 r.p.m. for 30 minutes at room temperature. Discard the supernantant.

12. Wash the pellet with 70% ethanol at room temperature and dry the pellet briefly under vacuum. Dissolve the pellet contai­ning plasmid DNA in 8 ml of Solution D.

Density gradient centrifugation:

1. For every ml of DNA solution, add 1 g CsCl and mix gently until all the salt dis­solves.

2. Add ethidium bromide to a final concen­tration of 600 ug/ml. The density of the final solution should be 1.55 g/ml.

3. Centrifuge at 18,000 r.p.m. for 15 min­utes at 15°C and carefully decant the supernatant.

4. Using a refractometer, check the refrac­tive index of the solution. The refractive index should be 1.3860. If it is not, a sat­urated solution of CsCl or distilled water can be used to increase or decrease the refractive index of the solution.

5. Pipette the solution carefully into Beckman ultra-clear tubes (5/8 × 3 in., No. 344085). Overlay with liquid paraffin and tighten the cap of the tube.

6. Spin the tubes at 48,000 r.p.m. for 36 hours at 15°C.

7. Remove the tubes carefully from the rotor and view the bands under UV light.

8. Collect the lower band which contains the plasmid using a sterile 5 ml syringe fitted with a 1.5 inch 19G needle.

9. Remove ethidium bromide by extracting with butanol a number of times (each time with twice the volume of the sample) until the last two extractions remain colorless.

10. Caesium chloride is removed by overnight dialysis at 4°C in excess (4 litres) of TE with at least three changes.

11. Examine the preparation by agarose gel electrophoresis.

Isolation of Plasmids by the Birnboim and Doly Method:

This is a mini-scale isolation procedure described by Birnboim and Doly (1979). This procedure is based on the differential behaviour of closed circular, open circular and linear DNA under alkaline conditions. High alkaline conditions bring about the separation of complementary strands of DNA in open cir­cular and linear duplexes but not in closed cir­cular DNA.

Therefore, exposing the crude lysate to a pH of 12 followed by neutralization results in the formation of insoluble aggregates of denatured DNA which can be removed by centrifugation. Plasmid DNA is then removed from the supernatant by ethanol precipitation. This method forms a rapid procedure for the isolation of plasmids and is suitable for trans­formation experiments and, sometimes, for restriction enzyme analysis.

Reagents:

Solution I:

Lysozyme – 10 mg

Glucose (20%, w/v) – 0.23 ml

Na₂EDTA (0.1 M) – 0.5 ml

Distilled water – 4.1 ml

Solution II:

Sodium hydroxide (2 M) – 1 ml

SDS (10%, w/v) – 1 ml

Distilled water – 8 ml

Solution III:

Sodium acetate, pH 4.8 (3 M) – 4.8 ml

Solution IV:

Sodium acetate (0.1 M) in Tris-HCl (0.5 M), pH 8.0

Solution V (TE):

Tris-HCl (10 mM) plus Na₂EDTA (1 mM)

Procedure:

1. Pipette 1.5 ml of an overnight bacterial culture into an Eppendorf tube and har­vest the cells by a brief centrifugation.

2. Resuspend in 0.1 ml of Solution I and leave on ice for 30 minutes.

3. Add 0.2 ml of Solution II, vortex gently and leave on ice for 5 minutes.

4. Add 0.15 ml of Solution III, mix by inver­sion and leave on ice for 45 to 60 min­utes.

5. Centrifuge for 5 minutes and transfer the supernatant as much as possible into another Eppend orf tube.

6. Add 1 ml cold ethanol (95%), mix and leave at -20°C for 30 minutes.

7. Collect the DNA by centrifuging in a microfuge for 10 minutes and resuspend the pellet in 0.1 ml solution IV.

8. Precipitate the DNA again using cold ethanol (95%) as in step 6.

9. Vacuum dry the sample to remove ethanol and dissolve it in 20 ul of solution V.

This method yields about 2 ug DNA.

Methodology # 2. Isolation of Chromosomal DNA:

Lymphatic filariasis is caused mainly by two species of parasitic nematodes, Brugia malayi and Wuchereria bancrofti. These par­asites are transmitted to the human host by mosquitoes (Nelson, 1979; Sim et al, 1986 a, b). To control a parasitic disease effectively, the biology of the organism, the nature and scope of the parasite problem must be assessed.

This has been hampered by difficul­ties encountered in detecting and identifying different filarial parasites. Genetic engineering techniques, however, have opened up an opportunity to do so. The following is an exer­cise in isolating genomic DNA from Brugia malayi microfilariae — a common parasite in parts of Asia. DNA of the cell coexists with RNA and other macromolecules.

Thus, during the purification steps, it is necessary to free the DNA from other cellular components. The cells are lysed using detergents, such as um dodecyl sulphate, and the proteins are removed by proteolytic digestion. The nucleic acids can then be separated by caesium chloride gradient centrifugation.

It is often difficult to isolate microfilariae free from contaminating blood cells of the hosts. A method of isolation using Percoll gra­dients that achieves this is described below.

Separation of Microfilariae from White Blood Cells:

This protocol describes the separation of Brugia malayi microfilariae from contamina­ting white blood cells.

Reagents:

1. TE buffer

2. RPMI medium

RPMI powder

(GIBCO Laboratories) – 10.4 g

HEPES – 5.94 g

Distilled water – 1000 ml

Sterilize by filtering through a 0.45 micron Millipore filter.

3. Percoll (Pharmacia) — polyvinylpyrroli­done coated colloidal silica particles.

Procedure:

1. Following the removal of adherent blood clots by washing in several changes in 10 mM phosphate buffered saline, pH 7.4, resuspend the microfi­lariae in 2 ml RPMI medium.

2. Prepare a Percoll gradient as follows:

(a) Pipette 2 ml Percoll into a Corex tube.

(b) Into another tube, mix 3 ml Percoll with 5 ml RPMI medium to give a final concentration of 0.6% Percoll.

(c) Layer the 0.6% Percoll carefully above the 2 ml Percoll cushion in the first tube to form two distinct Percoll layers.

3. Using a Pasteur pipette, carefully layer the microfilarial suspension on top of the Percoll gradient.

4. Centrifuge for 10 minutes at full speed in a bench centrifuge.

5. Remove the tube carefully from the centrifuge without disturbing the layers and stand it upright on the bench.

6. Remove the whitish band at the inter­face between the 0.6% and the undilut­ed Percoll layers using a pipette. This will contain most of the filarial worms.

7. Examine a drop of this preparation under the microscope.

8. If the preparation is still contaminated with white blood cells, repeat steps 2 to 7, but this time with 0.7% Percoll over the cushion. 0.7% Percoll can be pre­pared by mixing 3.5 ml Percoll with 5 ml RPMI medium.

9. Add five times the volume of RPMI medium to the final filarial preparation and spin down the worms at full speed in a bench centrifuge for 10 minutes.

10. Resuspend the pellet in 5 ml TE and spin again.

11. The worms can be stored at -70°C at this stage or resuspended in 200 ul of TE and used for the extraction of DNA.

Extraction of DNA:

Reagents:

Collagenase — 25 mg

SDS (10%, w/v) — 10 ml

Proteinase K — 10 ml

Caesium chloride — 20 g

TE buffer  — 4 litres

Chloroform: isoamyl alcohol (1: 0.04) — 10 ml

Procedure:

1. Add 25 mg of collagenase into 0.5 ml suspension of filarial parasite (about 100 mg wet weight) in TE and incubate at 37°C for 1 hour.

2. Add 50 ul of SDS (10%, w/v) and 450 ul of TE, mix and leave at 37°C for 30 minutes.

3. Weight in 10 mg of Proteinase K and incubate the mixture for 1 hour at 68°C.

4. Cool down to room temperature and add 2 ml chloroform: isoamyl alcohol mixture. Extract the undigested pro­teins with frequent agitation of the mix­ture for 20 minutes followed by removal of the organic layer.

5. Add 4 ml 4 × SSC to the aqueous layer and about 12 ml of ethanol (95%) and keep either at -70°C for 1 hour or -20°C overnight to precipitate the DNA.

6. Spin down the mixture at 18,000 r.p.m. for 15 minutes at 4°C. Remove ethanol completely by evacuating in a desiccator. Resuspend the pellets in a total of 2 ml TE. The sample at this stage can be used for genomic dot blotting after reprecipitating once more with twice the volume of ethanol and resuspending the pellet in 40 ul of TE.

7. The sample can be further purified by CsCl density gradient centrifugation. Mix the crude DNA preparation (1 ml) with 4 ml TE containing CsCl (1.25 g/ml) and transfer the solution into a Beckman Quick-Seal polyallomer tube (1/2 × 2 in., No. 342412). Fill up the tube, if necessary, with TE containing CsCl (1 g/ml), seal the tube and spin at 60,000 r.p.m. for 16 hours at 15°C.

8. Carefully remove the tube after the run, puncture the bottom of the tube and collect about 1 ml fractions in separate Eppendorf tubes. The sample contain­ing the DNA can be easily identified from the viscosity and the slow rate of flow of the drops. (If necessary, the fractions can be checked on a gel.)

9. Pool the fractions containing the DNA. Dialyse overnight at 4°C against an excess of TE buffer with at least three changes.

10. Concentrate the DNA by precipitating with ethanol (95%) in the cold, pelleting it and resuspending in 50 ul of TE.

Methodology # 3. Agarose Gel Electrophoresis:

Agarose gel electrophoresis has become a powerful and versatile tool in the investigation and characterization of DNA molecules. It is rapid, precise and inexpensive and requires only small amounts of DNA. Agarose is a polysaccharide extracted from various red algae. It is a polymer of repeating disaccharide units composed of β-D-galactopyranose and 3, 6 anhydro-L-galactose joined in a 1-3-β-glycosidic linkage.

Interchain hydrogen bonds are presumed to form the cross-links which lead to poly­merization. When DNA is subjected to agarose gel electrophoresis, it is forced to migrate through the interstices of this net­work toward the anode (due to its negatively charged phosphate residues) with a migration velocity determined by the molecular size of DNA, conformation of DNA, pore size of the gel (determined by the agarose concentration, see Table 3.1), voltage gradient applied to the gel and the electrophoretic buffer.

Agarose gels may be run horizontally as well as vertically. The horizontal gels are much simpler to set up and, for most purposes, give the resolution required. A number of well- designed horizontal gel tanks are available. The procedure described below is only suitable for the Agarose Gel Unit (Model HE-99) of Hoefer Scientific Instruments (San Francisco). However, the procedure can be easily modi­fied to suit any other apparatus.

Ethidium bromide is used in the detection of DNA bands on the gel. This dye interca­lates between the bases of the DNA molecule and gives DNA an orange fluorescence when irradiated with a short wavelength UV light source. Ethidium bromide slows the migra­tion of DNA by about 15 percent. Nonetheless, it is so convenient to monitor the progress of electrophoretic separation that ethidium bromide is usually included in the running buffer.

Reagents:

1. 10 × Gel buffer

The most commonly used buffer is Tris-Acetate-EDTA (TAE). Prepare a stock solution of the buffer as follows:

10 × Tris-Acetate-EDTA buffer (pH 8.2) Tris — 48.4 g

Sodium acetate (trihydrate) — 27.2 g

Na₂EDTA — 3.72 g

Distilled water — 1,000 ml

Adjust the pH to 8.2 with glacial acetic acid.

2. Running buffer

This is prepared by diluting 100 ml of 10 × Gel buffer to 1 litre in distilled water and adding 50 ul ethidium bromide. The final concentrations of the salts will be as follows:

Tris — 40 mM

Sodium acetate — 20 mM

Na₂EDTA — 1 mM

3. Ethidium bromide solution

Ethidium bromide — 10 mg

Distilled water — 1 ml

4. Agarose solution

Agarose NA (Pharmacia) — 0.8 g

Distilled water — 90 ml

10 × Gel buffer — 10 ml

5. Loading buffer

PEG 6000 — 1 g

Bromophenol blue — 2 mg

Distilled water — 10 ml

Procedure:

Casting gels:

1. Place the casting tray on a level surface and keep the running plate inside the casting tray.

2. Prepare the agarose solution as follows:

Weigh out 0.8 g agarose in a 200 ml bottle add 90 ml distilled water, mix and auto-clave for 10 minutes at 15 lbs per square inch. Leave the molten agarose to cool down to about 60°C and add 10 ml of 10 × gel buffer and 5 ul of ethidium bromide (10 mg/ml). Mix well and place the bottle in a water bath kept at 50°C until you are ready to pour the gel.

Warning: Do not Pour Agarose Hotter than 50°C or you will Risk Warping the Unit.

3. Pour the agarose into the casting tray. If agarose seeps under the running plate, it can be minimized by pushing down the handles of the running plate.

4. Place a comb across the rim of the casting tray to form sample wells. Adjust the height of the teeth so that they penetrate the gel but leave about 1 mm of gel between the teeth and the running plate.

5. Allow the gel to set for at least 30 min­utes.

6. Remove the comb carefully.

7. Lift the running plate out of the casting tray and peel off any gel adhering to the underside of the plate. Transfer the plate and the gel to the main unit.

Running the gel:

1. Fill each buffer chamber of the main unit with running buffer containing 1 ug per ml of ethidium bromide until the buffer reaches the upper surface of the gel, but not covering the gel itself. Fill the wells of the gel also with the same buffer.

2. Prepare the DNA samples as follows:

Pipette 5 ul of DNA (2 to 5 ug DNA) into an Eppendorf tube. Add 5 ul of loading buffer, mix and stand in a water bath at 70°C for 10 minutes. Cool the sample to room temperature.

3. Load the samples (10 ul) carefully using a Gilson pipette. (Note: The sample volume indicated above is for a well size of 7 × 1.5 mm. For other volumes, see Table 3.2).

4. Place the lid on the unit so that the cath­ode (black cord) is nearest the sample wells, since nucleic acid samples migrate towards the anode.

5. Connect the cables to the power supply. The power setting and the length of run will vary with requirements. Electrophoresis at low voltage, 20 volts per gel overnight (about 16 hours), is often suitable.

Detection of DNA:

DNA is normally visualized by fluores­cence of bound ethidium bromide.

Always use gloves to handle gels or buffers containing ethidium bromide which is a powerful mutagen.

1. Remove the gel with the running plate from the main unit and transfer the gel on to a black perspex sheet.

2. Observe the DNA bands fluorescing under short wavelength UV light inside a dark room.

Use goggles to prevent eye damage from the UV lamps.

3. Photograph the gel using type 667 films on a Polaroid camera fitted with an orange filter.

.

Calculation of the molecular size of DNA:

At low voltages, double-stranded linear DNA migrates at a rate which is inversely pro­portional to the log of its molecular size. Therefore by using standard DNA molecules of known size, the molecular size of any DNA may be obtained. The standard sizes of restric­tion fragments of bacteriophage lambda and pBR322 given in Table 3.3 can be used in size determinations.

The lambda DNA fragments are usually used as standard markers in agarose gel elec­trophoresis. The Sau3A fragments of pBR322 can be used in polyacrylamide gel electrophoresis for the analysis of smaller frag­ments of DNA.

An excellent review on electrophoresis is given by Manniatis (1982).

Recovery of DNA from agarose gels:

Some of the methods available for extracting DNA from gels are:

(a) Solubilization of DNA-containing agarose in a saturated solution of KI fol­lowed by hydroxyapatite column chro­matography;

(b) Phenol extraction of low-melting point agarose;

(c) With DEAE-cellulose paper.

Method (c) can also be used with polyacry­lamide gels and it supersedes other methods because of its simplicity, speed and high yield.

DEAE-cellulose paper method:

1. Cut DE81 DEAE-cellulose paper (Whatman) into pieces which are about 2-3 mm wider than the width of the DNA band to be extracted.

2. Soak these strips in 2.5 M NaCl for 4 hours, wash with distilled water and soak in running buffer.

3. Run a gel and locate the band of inte­rest using a short wavelength UV hand monitor.

4. Make a slit in the gel forward of the DNA band to be extracted and insert the DE81 paper.

5. Continue the electrophoresis until the ethidium bromide stained DNA migrates completely onto the paper. The migration of the band can be fol­lowed using the UV monitor.

6. Remove the paper from the gel. Elute the DNA by shredding the paper and incubating in TE-salt buffer (400 ul per 50 mm²) containing

Tris-HCl, pH 7.5 — 20 mM

Na₂EDTA, pH 7.5 — 1 mM

NaCl — 0.5 M

for 2 hours at 37°C.

7. Precipitate the DNA with 2 volumes of cold ethanol (95%) at -70°C for 1 hour.

8. Recover DNA by centrifuging in a microfuge for 15 minutes and dissol­ving in TE buffer.

The DNA fragments isolated by this method can be readily restricted and ligated, and successfully used as sequencing primers, too.

Methodology # 4. Restriction and Ligation:

Restriction of DNA:

DNA can be cut using restriction enzymes. Restriction enzymes are endonucleases which cleave phosphodiester bonds of double-strand­ed DNA at specific sites. There are 3 types (I, II and III) of restriction endonucleases. Of these, the type II enzymes, which recognize specific palinodromic sequences, are widely used in cutting DNA for gene cloning. There are over 600 restriction enzymes known today.

They are isolated from various species of bacteria and are named using a standard nomenclature. They are usually referred to by the first letter of the genus name, followed by the first two letters of the name of the bacterial species which form the source of the enzyme, as well as by a number indicating the sequence of detection of the enzyme in that bacterial strain. For example, EcoRI was the first restriction enzyme isolated from Escherichia coli RY13.

The type II enzymes form two classes:

(a) Enzymes which generate ‘sticky ends’ or ‘cohesive ends’; and

(b) Enzymes which generate ‘blunt ends’.

EcoRI recognizes the sequence,

5’….. GAATTC…… 3′

3’….. CTTAAG…… 5′

and it cleaves between G and A on both strands of DNA, giving ‘sticky ends’ as fol­lows:

Fragment I — Fragment II

5’….. G — AATTC…3′

3’….. CTTAA — G….. 5′

The overhanging sequences are comple­mentary to one another and, therefore, the fragments can readily base pair among them­selves. The enzyme Haelll (isolated from Haemophilus aegyptius), however, belongs to the second class. It recognizes a 4 base pair sequence:

5’….. GGCC…….. 3′

3’….. CCGG…….. 5′

and it cuts between G and C, giving ‘blunt ended’ fragments as follows:

Fragment I — Fragment II

5’….. GG — CC………. 3′

3’….. CC — GG………. 5′

In this case, the fragments cannot base pair.

Reaction conditions:

The reaction conditions for many type II enzymes are generally similar. The enzymes usually have quite broad pH optima (e.g. pH 6.5 to 8.6) and require Mg²⁺ ions. Conditions for Hind III digestion suit most enzymes:

DNA (in 10 mM Tris-HCl,

pH 8.0, 1 mM EDTA)

10 mM Tris-HCl, pH 7.5

10 mM MgCl₂

10 mM 2-mercaptoethanol

50 mM NaCl

Incubate with enzyme at 37°C for 1 hr.

Reactions can be stopped by adding excess EDTA (10 mM) or by heating the digests at 70°C for 10 minutes, followed by rapid cooling on ice. The heating step is nor­mally sufficient. It will denature the enzyme and will also dissociate cohesive ends of the fragments which will have annealed during the 37°C incubation.

Some restriction enzymes need either low salt (0 – 50 mM NaCl) or high salt (100 mM NaCl) concentrations.

There are also enzymes which require KCl instead of NaCl (Smal) or a higher tempera­ture of incubation (65°C, Tag]). It is always advisable to obtain the reaction conditions for each enzyme from the batch analysis sheet provided by the supplier before beginning with the digestions.

The enzymes are generally stored at -20°C.

Standard procedure for restric­tion of DNA:

1. Pipette the following into a sterile Eppendorf tube:

(a) DNA (up to 10 ug) — 10 ul

(b) 10 x Reaction buffer — 2 ul

(c) Restriction enzymes (10 units) — 2 ul

(d) Distilled water — 6 ul

Total volume — 20 ul

Mix the contents by tapping the tube and by centrifuging in a microfuge for about 30 seconds.

(10 × Reaction buffer contains the required components at 10-fold higher concentrations.)

2. Incubate the tube for 1 hour at 37°C.

3. Transfer the tube to a water bath kept at 70°C and leave it for 10 minutes.

4. Pipette 5 ul of the mixture into another Eppendorf tube and add 5 ul loading buffer to it. Electrophorese DNA on agarose gel and determine whether restriction has taken place.

Ligation of DNA:

Having described a method for cutting DNA molecules, we must consider a way in which DNA fragments can be joined to create recombinant molecules. The reaction of join­ing DNA molecules using the enzyme ligase is called ligation. There are two types of ligases:

(a) E. coli DNA ligase

(b) Phage T₄ encoded DNA ligase

Both E. coli and T₄ ligases seal single-stranded nicks between adjacent nucleotides in a duplex DNA chain by forming phosphodiester bonds. They differ in their co-factor requirements and the type of reactions they catalyse. The T4 enzyme requires ATP, whilst the E. coli enzyme requires NAD+. The T₄ enzyme can join both cohesive and blunt ended fragments whereas the other requires annealed cohesive ends.

The optimum temperature for ligation is 37°C but, at this temperature, the hydrogen bonds between the sticky ends are unstable. The ligation reactions are, therefore, done at temperatures ranging from 15°C down to 4°C with incubation times varying from 3 hours to 16 hours, respectively.

Standard Ligation-reaction:

DNA to be ligated with vector (150 ng) — 10 ul

Vector DNA (50 ng) — 5 ul

10 × Ligation buffer — 2 ul

T₄ DNA ligase

– For cohesive ends (0.03 U) — 1 ul

– For blunt ends (1 U) — 1 ul

Distilled water — 2 ul

Total volume — 20 ul

Incubate the mixture at 12°C-14°C for 16 hours.

10 × Ligation buffer contains:

Tris-HCl (1 M), pH 7.2 — 0.66 ml

MgCl₂ (1 M) — 0.1 ml

ATP — 2.4 mg

DTT — 15.4 mg

Water — 0.24 ml

Total — 1.0 ml

Methodology # 5. Transformation:

In 1928, Griffith introduced the term transformation when he observed that avirulent strains of Pneumococcus could be ‘trans-formed’ to virulent strains after contact with dead virulent cells. Avery et al later identified DNA as the active principle in transformation.

Transformation in bacteria is defined as a process of introducing exogenous DNA into a suitable bacterial host. Transformation of Escherichia coli by plasmid DNA is a proce­dure that is frequently used in gene cloning. E. coli, under natural conditions, will not take up and incorporate any exogenous DNA. However, E. coli cells can be made to do so simply by treating the cells with calcium chlo­ride.

Calcium chloride somehow makes the cells competent, resulting in yields of transformants as high as 10⁷ per microgram of DNA.

The transformation procedure described below is based on the method of Lederberg and Cohen (1974). The procedure involves (a) the preparation of competent cells, and (b) transformation by plasmid DNA.

Preparation of Competent Cells:

Reagents:

LG broth — 100 ml

MgCl₂ (0.1 M) — 25 ml

CaCl₂ (0.1 M) + glycerol (15%, v/v) — 5 ml

Procedure:

1. Inoculate 100 ml LG broth with an overnight culture of E. coli (1: 100).

2. Grow the culture at 37°C with shaking until the optical density at 600 nm reaches 0.60.

3. Spin down the cells at 4°C and resuspend in 200 ml MgCl₂ (0.1 M and cold).

4. Centrifuge the cells again at 4°C and resuspend the pellet, this time in 4 ml CaCl₂ (0.1 M) containing glycerol (15%, v/v).

5. Leave on ice for 90 minutes to gain maximum competency.

6. Store the cells at -70°C.

Transformation:

Reagents:

Saline/sodium citrate (1 × SSC)

Trisodium citrate — 44 mg

NaCl — 87 mg

Distilled water — 10 ml

Total — 10 ml

CaCl₂ (0.1 M) — 10 ml

LG broth — 150 ml

LG-agar + antibiotic plates — 5

LG broth — 100 ml

Agar — 1.6 g

Mix and autoclave for 20 minutes at 15 lbs/sq. in

Add the appropriate antibiotic (see Table 5.1 for amounts) prior to pouring the plates.

Procedure:

Prepare SSC: Ca²⁺ by mixing 3 ml SSC with 4 ml 0.1 M CaCl₂.

Place DNA (1 – 2 ug in 5 – 10 ul) in a 4 inch test tube kept on ice.

Add 100 ul of SSC: Ca²⁺.

Add 200 ul of competent cells, mix and stand on ice for 30 minutes.

Transfer the tube into a water bath kept at 42°C and incubates for 2 minutes.

1. Remove the tube to another water bath kept at 37°C. Add 0.3 ml rewarmed (37°C) LG broth and incubate for 1 hour.

2. Plate the cells on LG-agar containing the appropriate antibiotic and incubate at 37°C overnight.

3. Examine the transformants after 16 – 18 hours of incubation.

Note:

A control plate containing competent cells alone must be prepared in order to check the rate of spontaneous mutation of antibiotic resistance.

The common antibiotics used and their concentrations are given in Table 5.1.

Methodology # 6. Polyacrylamide Gel Electrophoresis:

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS- PAGE) is currently one of the most commonly used biochemical techniques for the character­ization and analysis of protein. It has been extensively used to determine the molecular weights of polypep­tide chains and to establish the homogeneity of protein preparations.

In addition, it has also proved to be a very valuable tool in the solubi­lization and fractionation of amphiphilic pro­teins. The applicability of SDS-PAGE has been greatly expanded and enhanced by the deve­lopment of two-dimensional electrophoresis and protein blotting (Western blot) techniques.

Principle:

Sodium dodecyl sulphate is a very strong anionic detergent. It is an amphipathic molecule that consists of a nonpolar hydrophobic region and a strong polar anio­nic group. In the presence of SDS and a reducing agent such as 2-mercaptoethanol, oligomeric proteins are dissociated into their constituent polypeptide chains. These poly­peptide chains were shown to migrate in SDS gels of the correct porosity according to their molecular weights, as given by the following expression:

R_f = A – B log₁₀ M.W. …10.1

where R_f = relative mobility

M.W. = molecular weight

A & B = constants for a particular expe­riment system

Thus, the size of the polypeptide chain(s) of a given protein can be determined by com­paring its electrophoretic mobility in SDS gels with the mobilities of marker proteins of known molecular weights.

Interaction of SDS with Proteins:

The empirically established relationship between electrophoretic mobility and polypep­tide chain molecular weight suggests a general mechanism for the interaction of SDS with proteins. The electrophoretic mobility of a polypeptide chain can be a function solely of its molecular weight only if

(a) The charge per unit mass (e/m) is approximately constant, and

(b) The hydrodynamic properties are a function only of the molecular length of the polypeptide chain.

To a first approximation, both criteria are met under the conditions of SDS-PAGE. SDS- binding studies of a variety of different proteins indicate that above a SDS monomer concen­tration of 8 × 10^-4M (0.02%, w/v), 1.4 g of SDS is bound per g of protein.

This means that the number of bound SDS molecules is approximately equal to half the number of amino acid residues present in the polypeptide chain. This high level of bind­ing and the constant binding ratio will in gene­ral “swamp out” the native (intrinsic) charge contribution of most proteins, and an approxi­mately constant negative net charge per unit mass will be obtained.

The resulting SDS-protein has been found to contain a high degree of secondary structure. Hydrodynamic and optical studies indicate that these complexes behave as rod-like particles (prolate ellipsoids) in which the particle length varies uniquely with the molecular weight of the polypeptide chain.

Based on this model of SDS-protein interaction, any chemical property of a given protein which would interfere with the average SDS binding, or cause deviations from the typical average charge per unit mass rela­tionship, may be expected to induce an abnor­mal behaviour of the protein upon SDS-PAGE.

Anomalies of this sort however, can usually be detected by carrying out the electrophoresis of both sample proteins and molecular weight markers at a number of different gel concen­trations and compiling a Ferguson plot for each polypeptide.

SDS-PAGE Buffer Systems:

There are numerous published buffer sys­tems for carrying out SDS-PAGE. They can be broadly classified either as continuous or dis­continuous (multiphasic) buffer systems, with variations coming mainly from differences in pH and/or addition of urea. In a continuous buffer system, the same buffer ions are present throughout the sample, gel and electrode vessels (although possibly at different concen­trations in each) at a constant pH.

An exam­ple of a commonly used continuous buffer sys­tem for SDS-PAGE is the phosphate buffer system of Weber & Osborn (1975). In con­trast, discontinuous buffer systems employ dif­ferent buffer ions in the gel and the electrode reservoirs. In addition, they also have disconti­nuities in buffer composition and pH as well as pore size. The SDS-Tris-glycine buffer system (Laemmli, 1970) is an example of a disconti­nuous buffer system for SDS-PAGE, and it is said to provide better resolution than the stan­dard SDS-phosphate system.

However, in a recent study on the use of continuous and dis­continuous buffer systems for SDS-PAGE, Lanzillo (1980) reported that some pro­teins exhibit different banding patterns in different buffer systems and, therefore, the choice of buffer systems may be more impor­tant than had been previously recognised.

Uniform Concentration versus Pore Gradient Polyacrylamide Gel:

SDS-PAGE can be carried out in either uni­form concentration or pore gradient polyacry­lamide gels. For any given uniform concentra­tion gel, it is important to realize that the linear relationship between log¹⁰ M.W. and relative mobility (Equation 10.1) is valid only over a lim­ited range of molecular weight. As a general rule, the following guidelines can be followed:

Buffer System Gel Concen. M.W. Range:

SDS-phosphate 15% — 12 – 45 kDa

10% — 15 – 70 kDa

With 10% and 5% gels, the upper limit is similar to the SDS-phosphate buffer system, but polypeptides with molecular weights less than 16 kDa and 60 kDa, respectively, have the same mobility as the buffer (dye) front.

Thus, it is important to choose an optimal gel concentration to effect fractionation and estimate the molecular weight of polypeptide chains for a given buffer system. In recent years, pore gradient polypeptide chains for a given buffer system. In recent years, pore gra­dient polyacrylamide gel has become more widely used for SDS-PAGE.

When compared with uniform concen­tration gel, pore gradient gel gives superior res­olution and sharper bands. In addition, it is also able to separate proteins of widely different sizes on a single gel. Thus this method can be used to estimate the molecular weights of polypeptide chains over a much wider molecu­lar weight range. The molecular weight values are given by the equation:

log₁₀ M.W. = a log₁₀T + b …10.2

where T is the total acrylamide concentration reached by the protein and a and b are the slope and intercept, respectively, of the linear regression line which is established from mea­surements of a set of standard proteins run in the same slab gel. Thus a plot of log₁₀ T should be linear for any shape (linear, concave and convex) of concentration gradients.

However, the calculation of T is simplified by using linear gradients, since to a first approxi­mation, a plot of log₁₀ M.W. versus log (migra­tion distance) is linear. As for uniform concentration gel, the range of molecular weights over which the above linear relationship holds depends on the gradient conditions chosen. Thus, linear relationships have been found for the following gradient gels and molecular weight ranges (Lambin and Fine, 1979):

(a) 5% – 20% T (%C = 2.6), molecular weight range, 14.3 kDa – 210 kDa

(b) 7% – 25% T (%C = 1.0), molecular weight range, 14.3 kDa – 330 kDa

(c) 3% – 30% T (%C = 8.4), molecular weight range, 13.0 kDa – 950 kDa

Note:

%T = (acrylamide + bisacrylamide)/vol- ume × 100

%C = bisacrylamide/(acrylamide + bi- sacrylamide) × 100

Low Molecular Weight Proteins and Peptides:

Low molecular weight polypeptides (<15 kDa) present a special problem in SDS-PAGE because they are not well-resolved in uniform concentration polyacrylamide gels, and typical plots of log¹⁰ M.W. versus relative mobility are found to change slope in this molecular weight range (The explanation for this seems to be that the intrinsic charge and conformation of small polypeptide chains are more important in determining their electrophorectic mobili­ties on SDS gels than large proteins).

However, it was reported that separation of these polypeptides was considerably improved by using highly cross-linked (i.e. high ratio of bisacrylamide to acrylamide) 12.5% polyacry­lamide gels, and by the inclusion of 8 M urea in the SDS-gel and sample buffers.

Recently, two urea-containing SDS-PAGE discontinuous buffer systems run on uniform concentration acrylamide gels applicable to the molecular weight range of 2.5 kDA – 90 kDa and 2.75 kDa – 27.5 kDa have been reported. They have been shown to pro­vide better resolution than the method of Swank and Munkres (1971).

On the other hand, three non-urea buffer systems run on pore gradient gels that are able to separate polypeptides over the molecular weight range of 1.5 kDa – 100 kDa, 2.0 kDa – 200 kDa and 1.3 kDa – 100 kDa have recently been published.

It is therefore apparent that these new buffer systems have greatly extended the lower molecular weight fractionation range of SDS- PAGE. This is an important development in protein chemistry since complex peptide mix­tures obtained from various proteolytic cleava­ges of proteins can now be separated by high resolution SDS-PAGE before being electroblotted onto glass fiber sheets for direct gas-phase microsequencing.

Methods of Detection:

There are numerous methods for detect­ing protein bands after SDS-PAGE. The most common method is undoubtedly the dye stain­ing technique. Coomassie blue is a very sensi­tive dye for this purpose; it is able to detect about 0.2 µg – 0.5 µg of protein per band. It forms electro­static bonds with the amino groups and non- covalent bonds with non-polar regions of pro­teins.

Recently, a highly sensitive silver stain has been introduced which is claimed to be up to 100 times more sensitive than Coomassie blue. Several fluorescent pro­teins labels have also been used to detect pro­teins. This method employs covalent labelling of proteins with fluorescent compounds such as dansyl chloride and fluorescamine prior to electrophoresis and detection of the fluores­cent bands after electrophoresis by UV light.

The main advantages of this method of detec­tion are that it is more sensitive than Coomassie blue (about 1 ng of protein can be detected with fluorescamine) and the progress of electrophoresis can be monitored by expos­ing the gels during a run to UV light in a dark­ened room.

Recently, visible labelling of proteins with dabsyl chloride for SDS-PAGE has also been reported. As these labelled proteins are colored (deep orange), they can be seen with the unaided eye, thus allowing the separation to be easily followed.

Proteins can also be biotinylated by reac­tion with biotinyl-N-hydroxysuccinimide and subsequently separated by SDS-PAGE before being electrophoretically transferred to nitro­cellulose paper for further characterization.

Finally, proteins which have been radiola­beled can be detected by autoradiography or liquid scintillation counting after SDS-PAGE. The methods that are commonly used to intro­duce radiolabels into isolated proteins are alkylation with [³H]- or [¹⁴C]-iodoacetate or iodoacetamide, or iodination with ¹³¹.

All these methods of detection (with the exception of radiolabelling) are nonspecific in nature in that they are applicable to all pro­teins. However, specific staining methods are also available. A good example of this is the periodic acid-Schiff (PAS) stain for detection of glycoproteins.

Experimental Section:

One of the most widely used multiphasic buffer systems for SDS-PAGE of proteins is that of Laemmli (1970). It is, in fact, just a modification of the method of Davis (1964) in which SDS is incorporated into both the gel and electrode buffers. In this discontinuous buffer system, the separating (resolving) and stacking gels are made up in Tris-HCl buffers at different concentrations and pHs while the electrode (tank) buffer is Tris-glycine.

During electrophoresis, the leading ion is chloride while the trailing ion is glycine. The experi­ment described below makes use of a uniform acrylamide gel concentration of 10.8% T, 2.6% C for the separating gel while the stack­ing gel concentration is 3.7% T, 2.6% C. The procedure is for electrophoretic apparatus (SE 200) from Hoefer Scientific Instruments. In addition, preparation of a 4.9% – 20.6% T (2.6% C) linear gradient gel for use with the SE 400 (Hoefer) is also given for comparison.

Reagents:

1. Acrylamide Monomer Solution (30.8% T, 2.6% C)

Acrylamide — 30 g

Bisacrylamide — 0.8 g

Water to — 100 ml

Filter through Buchner funnel with Whatman No. 1 filter paper. Store at 4°C in a dark bottle.

Caution:

Acrylamide and bisacrylamide are potent neurotoxic agents. Please wear gloves during handling and do not pipette by mouth.

2. Separating Gel Buffer (1.5 M Tris-HCl, pH 8.8)

Tris — 18.15 g

1M HCl — ~48 ml

Water to — 100 ml

Adjust pH to 8.8, if necessary.

Note:

89 ml of concentrated HC1 (11.3 M)/L will give ~1 M HCl.

3. Stacking Gel Buffer (0.5 M Tris-HCl, pH 6.8)

Tris — 6.0 g

Water — 40 ml

Titrate with 1 M HC1 (~ 48 ml) to pH 6.8, make up to 100 ml with water.

4. SDS Solution (10%, w/v)

SDS — 10 g

Water to — 100 ml

5. Ammonium Persulphate (Initiator) (10%, w/v)

Ammonium persulphate — 0.5 g

Water to — 5.0 ml

Use a freshly prepared solution.

6. Electrode Buffer (0.025 M Tris-0.192 M glycine, pH 8.3, 0.1% (w/v) SDS)

Tris — 3 g

Glycine — 4 g

10 ml of soln. 4

Water to — 1 L

7. Sample Buffer (2 x), (0.125 M Tris-HCl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 20% sucrose, 0.04% bromophenol blue)

Tris buffer — 2.5 ml of soln. 3

SDS — 4 ml of son. 4

2-mercaptoethanol — 1 ml

Sucrose — 2 g

Bromophenol blue (0.4%) — 1 ml

Water to — 10 ml

Store this solution frozen in 1 ml aliquots.

8. Staining Solution (0.1% Coomassie Blue R-250, 41.7% methanol, 16.7% acetic acid)

Coomassie Blue R-250 — 1.2 g

Methanol — 500 ml

Glacial acetic acid — 200 ml

Water — 500 ml

Filter through Whatman No. 1 filter paper.

Note:

Dissolve the dye in methanol first, and then add acid and water. It is best to use a freshly prepared solution.

9. Destaining Solution (30% methanol, 10% acetic acid)

Methanol — 300 ml

Glacial acetic acid — 100 ml

Water to — 1 L

Apparatus:

Although the following procedure is described for the SE 200 and SE 400 appara­tus from Hoefer Scientific Instruments (U.S.A.), any similar slab gel apparatus may be used for the same purpose. Before preparing the acrylamide mixtures, assemble the gel sandwich according to the manufacturer’s instructions, and check for any leaks with water.

Gel Preparation:

Uniform Concentration Gel:

The following is the recipe for a single 1.5 mm thick 10.8% T, 2.6% C separating gel, and 3.7% T, 2.6% C stacking gel to be run on the SE 200.

1. Mix the following solutions in separate fil­tration flasks:

Solution — Separating — Gel Stacking Gel

1 — 3.5 ml — 0.6 ml

2 — 2.5 ml — _

3 — _ 1.25 ml

4 — 0.1 ml — 0.05 ml

Water — 3.85 ml — 3.05 ml

Degas gel mixture with vacuum for 5 minutes with regular agitation, after which add

5 — 0.05 ml — 0.05 ml

TEMED — 10 µl — 5 µl

Final volume — 10.0 ml — 5.0 ml

2. Swirl gently to mix well, and transfer the separating gel solution to the gel sandwich with a plastic syringe fitted with a blunt needle to a height of 5.5 cm. Care must be taken not to introduce bubbles during this operation. Then gently overlay with about 100 pi of water-saturated butanol using a micropipette. It is important to work fairly rapidly as the gel mixture may partially polymerize before you have com­pleted these steps.

3. Allow the gel to polymerize at room tem­perature (about 15-30 minutes). When it has polymerized, there will be a clear line between the gel and the overlay. Remove the overlay with a syringe, and rinse the top of the gel with water or stacking gel buffer (0.125 M Tris-HCl, pH 6.8, 0.1% SDS).

4. Insert a comb into the top of the gel sand­wich, and introduce the pre-prepared stacking gel solution mixture with a syringe as in step 2 above. Remove any bubbles that are trapped in the ‘teeth’ of the comb by gently lifting or tapping the comb. After the stacking gel has polyme­rized, remove the comb carefully from the gel sandwich.

Pore Gradient Gel:

To prepare a pore gradient gel, additional apparatus such as a gradient mixer and a peri­staltic pump are required. The experimental set-up for preparing such a gel is schemati­cally shown below:

All the solutions required for preparing the pore gradient gel are similar to that used for the uniform concentration gel with the excep­tion of the stock monomer solution which has a concentration of 41.1% T 2.6% C

The gel formulation for preparing a 4.9% T to 20.6% T (2.6% C) gel to be run on the SE 400 is given below:

Stock Solution — 4.9% T, 2.6% C (A) — 20.6% T, 2.6% C (B)

Monomer — 1.8 ml — 7.5 ml

2 — 3.75 ml — 3.75 ml

4 — 0.15 ml — 0.15 ml

Sucrose — _ — 2.25 ml

Water to — 15.0 ml — 15.0 ml

Deaerate gel mixtures for 5 minutes with regular agitation, after which add:

5 — 60 ul — 60 ul

TEMED — 5 ul — 5 ul

Mix the solutions by swirling and pour them into the gradient mixer:

Solution A into chamber A and Solution B into chamber B. Open the valve between chamber A and B and start the stirrer and peri­staltic pump. Set the flow rate of the pump to about 3 ml/min for the delivery of the gel mix­ture to the gel sandwich. All the gel mixture (total volume of 30 ml) must be completely transferred in this process.

Overlay with about 200 ul of water-saturated butanol using a micropipette and leave the gel to polymerize (~1 hour). Immediately after this step, the gradient mixer and the peristaltic pump must be flushed out thoroughly with dis­tilled water to prevent acrylamide gelling in them. In the meantime, prepare the stacking gel mixture using the following formulation:

Solution — Stacking Gel (3.7% T, 2.6% C)

1 — 1.2 ml

3 — 2.5 ml

4 — 0.1 ml

Water — 6.1 ml

Deaerate under vacuum for 5 minutes with regular agitation, after which add:

5 — 0.1 ml

TEMED — 10 ul

Final volume — 10.0 ml

After the separating gel has polymerized, remove the gel overlay and rinse the top of the gel with distilled water or stacking gel buffer. Pour the stacking gel to within 5 mm of the top of the gel plates, and carefully insert the comb so as to avoid trapping any bubbles. Leave to polymerize, and start preparing sam­ples for electrophoresis (see below).

Sample Preparation:

Protein samples and molecular weight standards must be denatured and reduced completely before electrophoresis. This is usu­ally achieved by mixing the protein solution (preferably in a low ionic strength buffer such as 0.0625 M Tris-HCl, pH 6.8 i.e. a 1: 7 dilu­tion of stacking gel buffer without SDS) with an equal volume of 2 × sample buffer (solution 7) in an Eppendorf tube, and heating this mix­ture for 5 minutes at 95°C – 100°C. The treated sample is then chilled on ice before use. It can also be stored frozen at -20°C for future runs.

The final protein concentration should be about 1 mg/ml. The amount loaded for each lane will depend on the sensitivity of the stain­ing method as well as the complexity of the sample in question. For Coomassie blue stain­ing, a sample volume of 10 ul – 20 ul (corre­sponding to 10 ug – 20 ug of protein) is usu­ally satisfactory for most runs, especially for heterogeneous samples. For homogeneous samples on the other hand, a sample load of ~5 ug per lane is found to be sufficient. The recommended sample volume to load for molecular weight calibration kits manufactured by Pharmacia is 10 ul – 20 ul.

Assembly of Apparatus, Sample Loading and Electrophoresis:

1. SE 200:

Place the upper chamber gel sandwich assembly into the lower buffer chamber. Remove comb and rinse the wells with distilled water. Fill the sample wells and the upper and lower buffer chambers with electrode buffer (Solution 6). Each chamber requires about 75 ml. Use either a syringe or micropipette to load (underlay) each sample in a well.

When all the samples are loaded, place the safety lid on the unit and attach the leads to the power sup­ply. Run the gel at a constant current of 18 mA for about 2 hours (The starting voltage should be about 80 V at this setting, but will increase to about 200 V after 2 hours). Turn off the power supply, disconnect the leads and remove the safety lid of the apparatus.

Carefully remove the gel and transfer it to a plastic tray containing the staining solution (Solution 8). Leave for at least 4 hours (prefer­ably overnight) before pouring out the staining solution and replacing it with destaining solu­tion (Solution 9). Gentle agitation will usually speed up the destaining process.

2. SE 400:

Remove the gel sandwich from the gel casting stand and transfer it to the lower buffer compartment. Carefully remove the comb and rinse it with distilled water before filling each well with electrode buffer. Use either a syringe or micropipette to underlay each sample in a well.

After all the samples have been loaded, fir the upper buffer chamber to the top of the gel sandwich, taking care not to disturb the samples in the wells. Fill the upper and lower buffer chambers with 250 ml – 350 ml of elec­trode buffers (Solution 6). In the case of the upper buffer chamber, do not pour buffer directly over the slot in the bottom of the chamber as turbulence may disturb the sam­ples in the sample wells.

Attach the safety lid to the unit, and connect the leads to the power supply. Run the gel at a constant current of 25 mA for about 4 hours (the starting voltage should be approximately 140 V). At the end of the run, remove the gel carefully from the sandwich, and place it in staining solution (Solution 8) for at least 4 hours (preferably overnight). Transfer to destaining solution (Solution 9) to destain.

Estimation of Molecular Weights:

After destaining, measure the distance from the top of the separating gel to the pro­tein bands. Construct a standard curve by plot­ting log¹⁰ molecular weight against either the distance (for uniform concentration gels) or log¹⁰ distance (for linear gradient gels) migrat­ed by the marker polypeptides, from which the molecular weight of the unknown polypep­tide chain can be determined directly. The molecular weights of polypeptide chains obtained by this method are within 5% – 10% of those determined by other physicochemical techniques.

Methodology # 7. Two-Dimensional Electrophoresis:

Principle:

Two-dimensional (2D) electrophoresis is a powerful analytical technique, widely used to separate complex mixtures of proteins into many more components than is possible with one-dimensional electrophoresis. Commonly (though not necessarily) the mixture is first resolved on the basis of net charge, the second dimension subsequently resolving on the basis of molecular weight.

In this type of 2D elec­trophoresis (sometimes called ISO-DALT (Anderson and Anderson, 1978), the first dimension may consist of isoelectric focusing (IEF) or a variant of IEF known as non-equilibrium pH gradient gel electrophoresis (NEPHGE) (O’Farrell et al, 1977), and the second dimension is a standard discontinuous SDS gel system of the type described by Laemmli (1970). A recent review of this technique is given by Dunn and Burghes (1983).

The system is quite simple:

The first dimension is run in a tube gel which is then applied to the top of a polymerised slab gel. A small amount of agarose is usually used to complete physical contact between the two gels. The second dimension is run on a verti­cal slab. The proteins which had banded in the first gel migrate out of the tube and are further resolved in the slab gel. After the run the pro­teins appear as spots in the slab. (An alterna­tive system is to run the first dimension as a horizontal slab gel from which strips are cut, and applied to the second dimension gel.)

First Dimension:

(a) IEF:

In this technique the components of a mixture are separated on the basis of their iso­electric point (pi), which is the pH at which they possess no net electric charge. A pH gra­dient is formed in a gel between the cathode and the anode and the proteins migrate until they reach their pi, at which point migration ceases and the individual components concentrate into thin bands. IEF is, therefore, a steady state, or equilibrium, process.

The pH gradient is established by the electrochemical reactions occurring at the electrodes (H+O₃ ions are displaced towards the cathode), and is stabilised by the addition of amphoteric com­pounds known as carrier ampholytes. These highly mobile zwitterions have sharply defined pls and the mixture is chosen to have pi values evenly distributed over the desired pH range.

IEF can be carried out in either polyacry­lamide or agarose (low EEO) gels. Polyacrylamide has the advantage of low electroendosmosis (EEO), which is very important in IEF as electroendosmosis can cause the whole pH gradient to move towards the cath­ode (gradient drift), and thus disrupt the steady state process.

EEO is due to fixed charges on the gel matrix which attract counter ions, so that, in contrast to the bulk solution, the liquid layer close to the surface of the matrix will carry a net charge opposite to that of the sur­face. On application of an electric field, this charged liquid layer will migrate. When, as is commonly the case, carboxyl ions are the major ions contributing to EEO, at pHs above 5.5, a cathodic flow of water and solutes will take place and cause flooding at the cathode end of the gel.

The gel will also shrink around the pK of the carboxyl group. Other causes of gradient drift are atmospheric CO₂ and charged impurities in the reagents. Gradient drift can be significant when running IEF in gel rods, in particular for the O’Farrell technique where the polyacrylamide gel contains 8 M Urea and Nonidet NP-40 for sample solubi­lization. Urea should be deionised prior to use. IEF gels containing urea require longer focus­ing times to reach equilibrium.

While polyacrylamide is the matrix of choice for IEF, it has the disadvantage of being impermeable to very large proteins, even at low gel concentrations. Low EEO agarose can be used for IEF of such proteins.

At the completion of the IEF run, the actu­al pH gradient generated can be determined in two ways. One way is to run a duplicate gel with a mixture of standard proteins of known pls; a pH gradient curve can be constructed from the positions of the known proteins.

Another way to determine the pH gradient is to cut a duplicate gel into slices, and place the slices in a small volume of dilute salt. On incu­bation, the ampholytes diffuse out of the gel slices and the pH of each slice can be mea­sured on a pH meter. (Alternatively, a surface electrode can be used to measure the pH directly on the gel.) It should be noted, howev­er, that there are problems with both these methods when IEF gels are run in the pres­ence of urea.

Many of the commercially avail­able standards do not give acceptable results in urea, due to the formation of multiple spots and removal of coloured prosthetic groups. Measurement of the pH of gel slices where the gel was run in the presence of urea can also lead to large errors.

The approach to equilibrium of the gradi­ent is asymptomatic and the number of volt hours (Vh) required for a protein to focus can vary with the conditions used and the proteins under investigation. The number of Vh neces­sary for equilibrium varies with the square of the length of the gel.

Therefore, for repro­ducible conditions, it is necessary to carefully control both Vh and gel length. Various meth­ods can be used to control equilibrium: e.g. coincidence of bands when samples are co-migrated from the anode and the cathode (best done on a horizontal slab gel), and con­stancy of pattern over long focusing times.

(b) NEPHGE:

This technique is similar to IEF except that the proteins do not reach their isoelectric point as equilibrium is not reached. The advantage of this technique is that it can resolve both acidic and basic proteins whereas in IEF there is frequently a loss of basic pro­teins due to cathodic drift. For NEPHGE, pro­teins are loaded at the acidic end and elec­trophoresis is allowed to proceed for only a relatively short time; the proteins are separat­ed in the presence of a rapidly forming pH gradient.

Whereas an estimate of the pls of the indi­vidual components of the mixture cannot be obtained, non-equilibrium techniques can be useful, as it is the separation of proteins that is of interest and not equilibrium per se. However the non-equilibrium gel may not show optimal resolution of small charge differ­ences between proteins, or be as reproducible as IEF. Reproducibility is very sensitive to experimental conditions.

Second Dimension:

The second dimension is a discontinuous sodium dodecyl sulphate (SDS)-polyacrylamide gel, where separation is based on molecular weight. Most proteins, when heated with the anionic detergent SDS, in the presence of reducing agents, are unfolded, and bind a con­stant amount of SDS (about 1.4 g per g of protein).

Under these conditions the charge: mass ratio of each species is relatively constant, and the electrophoretic mobility of a protein is inversely proportional to the log of its molecular weight (Weber and Osborn, 1969). Comparison with the mobility of known standards thus allows determination of the molecular weights of unknown proteins.

Anomalous migration does occur with some proteins under these conditions. For example, carbohydrate moieties do not bind SDS, so glycosylated proteins may migrate more slowly than non-glycosylated proteins of the same molecular weight. Non-reduced pro­teins may also migrate anomalously, as May some proteins which are not fully unfolded by the SDS treatment.

A number of buffer systems have been developed for SDS-polyacrylamide gel elec­trophoresis (SDS-PAGE), the most commonly used being that described by Laemmli (1970). This is a discontinuous system, i.e. two gel lay­ers are used: a short, large pore gel called the stacking gel, is layered on top of a separating gel.

The buffer components are chosen such that the mobility of the proteins is intermedi­ate between the mobility of the buffer ion in the stacking gel (leading ion) and the mobility of the buffer ion in the upper tank (trailing ion); the anionic complexes of SDS and protein are thus concentrated into a very thin zone between the leading ion and the trailing ion.

The separating gel can be a single pore size which is chosen to give best resolution of the proteins of interest; high concentrations of acrylamide give better separation of low molecular weight proteins, whereas low con­centrations of acrylamide are used for the sep­aration of high molecular weight proteins.

If the proteins of interest are very heterogeneous with respect to size, it may be preferable to use a gradient gel as the separating gel, with either a linear or exponential gradient. This is formed using a concentration gradient of acry­lamide which produces a gel of decreasing pore size. When proteins are electrophoresed in this gel, they eventually reach a position in the gel where the pore size is sufficiently small to reduce their mobility to zero.

In many cases a stacking gel is not neces­sary as the IEF gel acts as its own stacker.

Equilibration of First Dimension Gel:

This procedure, while apparently neces­sary to avoid streaking of high molecular weight proteins, can nevertheless result in considerable loss of protein, especially if extended times are used. In addition, loss of resolution can occur due to diffusion of the protein bands. Therefore, a relatively short equilibration is recommended. Some investi­gators omit this step entirely, relying on equi­libration with SDS occurring in the stacking gel.

Reagents:

(Note: Urea should be ultra-pure grade, or should be deionised prior to use.)

1. Sample buffer (9.5 M urea, 2% NP-40, 2% ampholyte, 5% 2-mercaptoethanol)

Urea — 28.5 g

10% NP-40 — 10 ml

Ampholines pH range 5-7 — 1.6 ml

Ampholines pH range 3.5 – 10 — 0.4 ml

2-mercaptoethanol — 2.5 ml

Make up to 50 ml.

2. Gel overlay solution (8 M Urea)

4.8 g urea/10 ml

3. Sample overlay solution

To 100 pi sample buffer, add 20 µl deionised distilled water.

4. Equilibration buffer (10% glycerol, 2.3% SDS, 5% 2-mercaptoethanol, 62 mM Tris, pH 6.8)

Glycerol — 50 ml

SDS — 11.25 g

10 x stacking buffer — 25 ml

2-mercaptoethanol — 25 ml

Make up to 500 ml.

5. NaOH cathode soltuion (0.02 M NaOH)

(Note: It is advantageous to prepare this sev­eral hours before it is needed. Boil for about 5 minutes, de-gas and allow to cool.)

6. Phosphoric acid anode solution (10 mM phosphoric acid)

0.7 ml orthophosphoric acid in 1 litre deionised distilled water.

7. Stock acrylamide (for IEF and NEPHGE gels)

acrylamide — 28.4 g

N, N’-methylene bis-acrylamide — 1.6 g

Make up to 100 ml.

8. 10% (w/v) ammonium persulphate (APS)

(Note: Prepare fresh, daily).

9. 10% Nonidet P-40 (NP-40)

10% (w/v) in deionised distilled water.

10. IEF gel solution

Urea — 5.5 g

Ampholines pH range 5-7 — 0.4 ml

Ampholines pH range 3.5 – 10 0.1 ml

Acrylamide stock solution — 1.3 ml

Water — 1.97 ml

10% NP-40 — 2.0 ml

10% APS — 14 ul

TEMED — 9 ul

11. NEPHGE gel solution

As for IEF gels, except use 0.5 ml ampholines pH range 3.5 – 10 and omit pH range 5-7 ampholines.

12. Stock acrylamide for SDS gels

Acrylamide — 58 g

N, N’- bis-methylene acrylamide 2 g Make up to 200 ml.

13.5 × Separating buffer (pH 8.8)

Tris — 225.06 g

SDS — 5.0 g

Adjust pH with HCl.

Make up to 1 liter.

14.10 × Stack buffer (pH 6.8)

Tris — 75.69 g

SDS — 5.0 g

Adjust pH with HCl.

Make up to 500 ml.

15. Agarose (1% agarose in equilibration buffer)

1 g agarose/100 ml equilibration buffer.

16. SDS separating gel solution (8.5% acry­lamide)

(This is sufficient for 2 gels approximately 16 cm × 12 cm × 1.5 mm)

Stock acrylamide — 17.0 ml

5 × separating buffer — 12.0 ml

Water — 31.0 ml

10% APS — 200 ul

TEMED — 60 ul

17. SDS stacking gel solution (3% acrylamide)

Stock acrylamide — 2 ml

10 × stacking buffer — 2 ml

Water — 16 ml

10% APS — 60 ul

TEMED — 24 ul

18. SDS tank buffer (10 × concentrated)

Tris — 30.28 g

Glycine — 144.14 g

SDS — 10 g

Make up to 1 litre.

Dilute 1: 10 for use.

19.0.1% Bromophenol blue

1 mg/ml in distilled water.

20. Molecular weight standards

Note:

Molecular weight standards are only accurate when reduced. They should be boiled in SDS sample buffer, and can be stored as frozen aliquots at -70°C.

When dealing with a heterogeneous unknown sample, it is best to use a mix­ture of high and low molecular weight standards.

21. pl markers

As mentioned in the introduction, most commercially available standards do not give reliable results in urea IEF gels. They are, how­ever, useful for visual monitoring of the gels during a run. They are dissolved in IEF sample buffer just before use. (Alternatively, a “charge train” of carbamylated protein may be gener­ated by heating in the presence of urea. Such mixtures are useful for com­parison of one gel with another.)

Silver Staining Solutions:

Note: Use double distilled water for all these solutions.

1. Fixative

50% methanol

10% acetic acid

2. Formaldehyde (0.1% (w/v) formaldehyde) 0.5 ml 40% formaldehyde to 200 ml water.

3. Diamine solution

(Note: Prepare prior to use)

Solution A: 5.25 ml water

45 ul 1 M NaOH

0.75 ml methylamine (co­mmercial grade, 40%)

Solution B: 20% (w/v) silver nitrate Add approximately 4 ml Solution A to 2 ml Solution B, or until brown precip­itate just clears.

Add 44 ml water.

4. Concentrated HCl

5. Developer

5 ml 0.5% citric acid

0.5 ml formaldehyde

Make up to 500 ml with water.

Protocol:

Caution:

Acrylamide and bis-acrylamide are neurotoxins which can be absorbed through the skin. Wear gloves while handling, preferably weigh out solids in a fume hood, and do not pipette solutions by mouth.

Sample preparation:

(This can be carried out while gels are poly­merising.)

Note: It is important that the salt concentra­tion of the sample is kept low and that the samples are not heated once IEF sample buffer has been added.

For immunprecipitates:

Resuspend the pellet in about 50 ul IEF sample buffer. Centrifuge in microfuge for 5 minutes.

For whole extracts:

If volume is x ul, add x ul IEF sample buffer and x mg urea. Ensure that all urea has dissolved.

IEF Gels:

1. Glass tubes should be thoroughly clean and dry. Seal the ends of the tubes with two layers of parafilm and insert vertically into stand. Make a mark on each tube 12.5 cm (or described length) from bottom with a marking pen.

2_: Mix the monomer solution in a side-arm flask, omitting the NP-40, TEMED and ammonium persulphate. Ensure that all urea is dissolved before continuing. Gentle warming may be necessary to dissolve all the urea.

3. Deaerate the solution with vacuum for about 5 minutes with regular stirring.

4. Add the NP-40. Swirl gently to mix.

5. Add the TEMED and ammonium persul­phate. Gently swirl to mix.

6. Using either a Pasteur pipette or a syringe and needle, fill each tube to the mark with the solution, being careful not to introduce bub­bles.

7. Gently overlay the gel solution with over­lay solution (about 2-3 mm).

8. When the gel has polymerised there will be a clear line between the gel and the over­lay. Remove the overlay solution with a syringe, or by gently flicking the tubes. Apply about 20 ul IEF sample buffer and leave gels to stand for 1 – 2 hours.

9. Remove parafilm from bottom of tubes and insert tubes into upper buffer chamber. (Do not force the tubes through the grommets or they may break). Tubes should extend about 2 mm above the grommets. Insert the upper chamber into the lower chamber containing the phosphoric acid anode buffer. Add NaOH cathode buffer to upper tank.

10. Pre-run the gels for 15 minutes at 200 V, then 30 minutes at 300 V, then 30 minutes at 400 V.

11. Turn off power; wait for voltage to fall to zero.

12. Remove cathode buffer and overlay from the gels.

13. Load samples onto top of gels, being care­ful not to introduce bubbles. Overlay with 10 ul sample overlay solution, then with NaOH cathode buffer. Refill upper chamber with NaOH cathode buffer.

14. Run at 300 V – 400 V for a total of 4,800 Vh, followed by 800 V for 1 hour.

15. Remove tubes and shake out excess buffer. Extrude gels into screw capped vials half-filled with equilibration buffer. To remove gels from tubes, manual pressure using a rub­ber teat, or a syringe with a piece of plastic tubing attached, is often sufficient.

If gels are hard to extrude, they can be “rimmed” by inserting a blunt thin needle and running it around the edge between the gel and the glass. Breaking of the gels during extrusion may be caused by extruding the gel too fast. Overloading the gels with protein can weaken them and may also lead to breakage during extrusion.

16. The gels are equilibrated for about 30 minutes at which point they may be loaded on the second dimension gel, or frozen for later use. (Note: Frozen gels are somewhat more fragile to handle. It is best to freeze rapidly in an ethanol bath, to avoid formation of microbubbles in the gel.)

NEPHGE Gels:

1. Set up gel tubes and pour gels as for IEF (Note: Different solutions).

2. Load polymerised gels into gel chamber as for IEF gels, but Note for NEPHGE the upper chamber contains the anode (positive) buffer.

3. Overlay with 100 ul overlay solution then with phosphoric acid anode solution. Fill chambers with electrode buffers.

4. Run at 550 V for 5 hours (constant volt­age).

5. Extrude gels and treat as for IEF gels.

SDS Slab Gels:

1. Assemble gel mould, ensuring that all seals are leakproof and that the plates are level. (Note: Some systems, particularly “home­made” devices, require that the bottom and sides of the gel plates be sealed with agarose. To do this, make up a 1% agarose solution containing 0.1% SDS, store in a 55°C – 60°C oven. Pre-warm the assembled plates and a Pasteur pipette. Seal the sides and bottom of the gel plate assembly with the agarose solu­tion and allow to set at room temperature).

2. Make up both the gel solutions, omitting the TEMED and ammonium persulphate. (Note: If the gel is to be silver-stained it is a good idea to filter the solutions at this stage. Filter through a Millipore 0.22 micron filter or equivalent). De-gas the separating gel solution with vacuum for about 5 minutes.

3. Add the TEMED and ammonium persul­phate to the separating gel solution, swirl to mix. Add gel solution to gel mould to within about 4 cm of the notch.

4. Overlay the gel solution with 1-2 mm of water-saturated butanol or 0.1% SDS being careful not to mix the layers.

5. Allow the gel to polymerise at room tem­perature. This should take about 45 minutes.

6. Remove overlay (if butanol was used, rinse top of gels with 1 x stack buffer).

7. Add TEMED and ammonium persulphate to stacking gel solution, swirl to mix.

8. Pour stacking gel to within 2-3 mm of the top of the glass plate. Insert a single reference well comb, taking care not to trap any bubbles.

9. Set up agarose solution in boiling water bath.

10. When stack gel has set, remove comb and rinse the top of the gel with tank buffer. (Again, depending on the type of apparatus you are using, you may need to load gels into tanks at this stage, or it may be better to do this when the first dimension gels have been loaded).

11. Pour off sample buffer from equilibrated IEF or NEPHGE gels, preferably onto stainless steel mesh over a beaker. If frozen, these should be thawed for about 15 minutes before required.

12. Lay tube gels onto a piece of parafilm. Identify cathode end (usually uneven and slightly shrunken).

13. Slide the tube gel into place on top of the slab gel orienting so that the cathode is to the right. If necessary, smooth into place with a spatula and eliminate any air bubbles. Overlay with 0.5 ml – 1 ml melted agarose solution. (An alternative procedure can be used if it is possible to warm the second dimension gel to 30°C – 40°C. Warm the slab gel, add melted agarose first, and then quickly slide in the tube gel). Do not move the gel for a few minutes while the agarose sets.

14. If the gels have not been loaded into the tanks, do so, then load the molecular weight standards (5 ul per well).

15. Add tank buffer to both chambers. To upper chamber, add 2 drops of 0.1% bro- mophenol blue.

16. Electrophorese at 25 mA/gel (constant current) until sample has migrated through the stacking gel, then 35 mA/gel until bromophenol dye marker reaches a few mm from the bottom of the gel.

17. At end of run, remove slab gel from the plates (open plates with a large spatula) and place in fixative. The tube gel may be discar­ded.

Silver Staining:

Note: Wear gloves. Gel containers must be very clean.

1. Soak gel for 1 hour in fixative.

2. Wash gel at 60°C in two changes of water, the first wash for 10 minutes, the sec­ond for 20 minutes.

3. Incubate gel at 60°C for 30 minutes in 0.1% formaldehyde solution, 200 ml/gel.

4. Cool gel in water for 10 minutes at room temperature.

5. Shake gel at room temperature for 10 minutes in diamine solution. (Reminder: Prepare just before use).

6. Discard the diamine solution into a beaker and quickly rinse gel in two changes of water.

Note: The discarded diamine solution is potentially explosive. Neutralize immediately with hydrochloric acid.

7. Develop gel with two changes of developer until staining has reached the point required. Wash gel with several changes of water.

Destaining:

This is only required if development of gel has proceeded too far.

Solution A: Dissolve 11.1 g NaCl and 11.1 g cupric sulphate in 285 ml distilled water. Add ammonia solution (25%) until precipitate clears to give a deep blue solution (final vol­ume is about 300 ml).

Solution B: Dissolve 44 g of sodium thiosul- phage pentahydrate in 85 ml distilled water (final volume is about 100 ml).

Diluted Kodak Hypo-clearing agent: 20 g in 800 ml distilled water.

1. Mix 15 ml solution A with 5 ml solution B, dilute with 40 ml water.

2. Incubate gel in this solution until a little less than required destaining has occurred.

3. Quickly rinse gel in water and incubate in 100 ml diluted Kodak Hypo-clearing agent for 30 minutes.

4. Wash gel for 10, 20 and 30 minutes in three changes of water.

Alternatives:

1. Although silver staining is more sensitive, the gels may also be stained for protein with Coomassie Blue: Immerse gel directly into a solution of 0.1% Coomassie Brilliant Blue R- 250 in 20% methanol/10% acetic acid. Stain at least 2 hours. Destain with 40% methanol/10% acetic acid, then with 5% methanol/10% acetic acid. The staining solu­tion may be reused several times.

2. If the sample has been metabolically or otherwise radiolabeled (e.g. with ¹²⁵I), the gel may be dried and autoradiographed.

3. The proteins separated on the two-dimen­sional gel may be transferred to nitrocellulose by the Western blot ‘procedure, and probed with specific reagents (e.g. antibodies, lectins).

Methodology # 8. Immunoelectrophoresis:

Principle:

Quantitative estimation of proteins by pre­cipitation with corresponding antibodies has been of fundamental importance in the deve­lopment of immunochemistry. A simple, quick and reproducible method known as electroim- munoassay (EIA), “rocket” Immunoelec­trophoresis or electroimmunodiffusion was first described by Laurell (1966).

In this method, a monospecific antiserum is first incorporated into the agarose gel at a uniform concentration and the standard antigens and test samples are introduced into wells which are punched out along one side of the agarose gel plate. A fast precipitation reaction between the antibody and antigen in the agarose gel is then achieved by the application of an electric field.

As the antigen (Ag) starts to migrate from the well into the gel containing the antibody (Ab), a precipitation zone like an ascending rocket is formed when a critical Ag/Ab ratio is reached. Initially, the complex formed between antigen and antibody is soluble since the concentration of antigen is in excess.

The successive consumption of antigen in the for­mation of the side lines results in convergence of the precipitation lines to a peak. The final position of the precipitation frontier of each antigen at a given antibody concentration in the agarose gel would vary with the amount of the antigen applied. The height of the rocket is directly proportional to the concentration of the antigen.

In general, the electrophoresis is carried out at a chosen pH value at which the antigen will migrate and the electrophoretic mobility of the antibody is very low or remains essentially stationary during electrophoresis.

If the electrophoretic mobility of the anti­gen at the chosen pH is low, such as (3- and y- globulin at pH 8.6, a slow development of blunt precipitate peaks or formation of oval precipitate lines would occur which is inappro­priate for quantitation. In this case, the isoelec­tric point of the antigen can be decreased by acetylation or carbamylation or increased by treatment with carbodiimide and a nucleophile.

This would increase the net charge and the electrophoretic mobility of the antigen, but only cause a slight decrease in the antibody affinity. The quantitative rocket immunoelectrophoretic analysis can also be used to determine the concentration of pro­teins solubilized with sodium dodecyl sulphate or Triton X-100 in the presence of polyethy­lene glycol.

Equipment:

1. Power source

2. Electrophoresis chamber

3. Glass plate or plastic polyester film

4. Gel punch template

5. Gel punch

6. Micropipette.

Reagents:

1. Barbital buffer (pH 8.6)

0.075 M sodium barbital buffer

0.002 M calcium lactate

0.02% (w/v) sodium azide

The presence of calcium lactate in the gel containing the antibody has been found to enhance precipitation and sodium azide is used as a preservative.

2. 2% Agarose gel solution

1.0 g of agarose (< 0.2% sulphate con­tent) is dissolved in 50 ml of barbital buffer, pH 8.6. The mixture is placed in a flask and heated in a boiling water bath with constant stirring until the agarose has completely dis­solved. It is then cooled to 50°C – 55°C.

3. Antiserum

IgG fraction of the antiserum against a specific antigen.

4. Washing solution

0.1 M NaCl

5. Staining solution

0.5 g of Coomassie Brilliant Blue R250 is dissolved in 200 ml of ethanol: acetic acid: water (90: 20: 90). This staining solution can be used several times.

6. Destaining solution

The same as staining solution but without Coomassie Brilliant Blue.

Procedure:

Electrophoretic Run:

1. 50 ul antibody in 6 ml barbital buffer warmed to 50°C – 55°C is mixed with 6 ml of 2% agarose solution in barbital buffer previ­ously prepared and cooled to 50°C – 55°C. To avoid air bubbles, the mixing is done by turning the test tube end over end a few times.

2. A clean and dry glass plate or polyester film (84 × 94 × 1 mm) is placed on a horizon­tal table properly levelled.

3. The molten gel containing the antibody is quickly poured onto the middle of the plate and dispersed over the entire support surface by means of a pipette tip or spatula to ensure a smooth uniform thickness. Let the gel set at room temperature for 30 minutes. (In quanti­tative work the antibody in the gel must be evenly distributed and the gel must be of uni­form thickness throughout the whole surface of the plate.)

4. Using a gel punch and gel punch tem­plate, a linear row of application wells are punched out from the gel with centres at least 5 mm apart between the two neighbouring wells. (A well with 3 mm in diameter can accommodate a 10 ul sample.)

5. Transfer the gel plate to an electrophore­sis chamber.

6. 10 ul of test samples and standards with a concentration range of 1 ug – 5 ug in barbital buffer are quickly introduced into the respec­tive wells. If a micropipette is used, the pipette tip should lightly rest against the plate during delivery of the sample. This would give a slightly concave fluid surface and eliminate the risk of antigen spreading around the well. The time taken to introduce the first and the last samples should not exceed 5 minutes so as to avoid effects of excessive diffusion and spread of the samples.

7. Connect the electrophoresis cells and electrode buffer vessels by means of two wicks of Whatman 3 MM filter paper, pre-wetted with barbital buffer, as conducting bridges.

8. A power supply is connected. A maximum of 15 V/cm may be applied if the elec­trophoresis apparatus has a cooling system or electrophoresis is carried out in the cold room. Electrophoresis time is dependent on molecu­lar size, surface charge of antigen and anti­body and the number of antibody combining sites.

Drying:

After electrophoresis, the gel is removed from the electrophoresis apparatus and its entire surface covered with a single layer of fil­ter paper pre-wetted with distilled water, (care being taken to avoid inclusion of any air bub­bles). A few layers of soft blotting paper are placed over the covered gel and the gel is then lightly pressed for 10 – 15 minutes.

The pressed gel is washed with 0.1 M NaCl for at least 15 minutes. Gel with high concentrations of antibody or unfractionated antiserum may have to be washed overnight in the saline solution.

Finally the gel is washed a few times with distilled water in order to remove the NaCl and to prevent the formation of salt crystals in the dried gel. The gel is then dried to a fine film with a hair dryer.

Staining and Destaining:

When the gel is dried completely, it is placed in the staining solution for 5 – 10 min­utes. The gel is destained with the destaining solution until the background is clear. Finally the gel is dried with a hair dryer.

Estimation of Peak Height and Calculation of Antigen Concen­tration:

The plate with the dried gel film facing downward is placed on a graph paper and each precipitate peak height is measured from the center of each well to the rocket tip. A standard curve is constructed. Since the height of the rocket bears a linear relationship to anti­gen concentration, the concentrations of the test samples can be read directly from the standard curve. For precise quantitative deter­mination, rocket height must be proportional to antigen concentration and the morphology of rockets from both samples and standards must be identical.