In this article we will discuss about the Paper and Gel Electrophoresis of Serum Protein.
Paper Electrophoresis of Serum Protein:
Introduction:
Electrophoresis is a method by which electrically charged particles of a mixture of substances are separated under an electric field. The positively charged particles migrate towards cathode, and the negatively charged particles towards anode.
The rate of migration depends on the number of charges of each particle carries. As a result of different rates of migration, a mixture of proteins (such as plasma) can be separated into a number of fractions having similar mobility.
Tiselius (1937) first performed electrophoresis in a fluid electrolyte. In this method, the differently charged particles moved inside the buffer. This method was called “moving boundary electrophoresis”. Few workers only used this method because of the costly and complicated apparatus.
Consden, Gordon, and Martin (1946) introduced filter paper to hold the electrolyte in the electrophoretic technique. Filter paper was the first anti-convection medium to be widely used. Later on, other media such as cellulose acetate, agar gel, starch gel, starch block and acrylamide gel have been used in place of paper with better results.
Principle:
A small amount of serum is applied on a Whatmann filter paper No. 1 strip previously soaked in buffer solution, the two ends of the strip dip in two compartments of an electrophoresis tank containing the buffer solution. A direct current is applied to the ends of the paper for a desired period.
During this time different fractions of the protein mixture separate into bands depending upon the amount of charge, the shape and size of the protein particles. After that electric current is disconnected, paper is removed from the tank, dried and stained with a protein stain to demonstrate different protein bands.
The fastest band is albumin, behind it are α1-globulin, α2-globulin, β-globulin and γ-globulin (shown in fig. 46.5). The stained fractions are eluted in suitable solvent and the eluted colour is measured in a colorimeter.
Two types of electrophoretic tanks are available:
a. Vertical and
b. Horizontal.
a. In the vertical type, there are five compartments. The electrodes are present in two outermost compartments, which is connected to adjoining compartments by wicks. By this arrangement the pH change occurring in the electrode compartment is prevented from affecting the adjoining compartments in which the ends of the paper dip.
In the middle, there is a central compartment. The arrangement is shown in Fig. 46.4. All compartments excepting the central one are filled by buffer.
b. In the horizontal type, paper lies in horizontal position supported by strings. The paper ends dip in two compartments on the two sides of the tank. These compartments are connected by means of wicks to adjoining compartments containing electrodes. Buffer is kept in all compartments to equal levels.
Procedure:
Pour sufficient buffer into the four compartments up to the same level, leaving the central compartment empty, cut Whatmann No. 1. paper into strips of 35 cm long and 5 cm wide. Mark the centre of the strip with a pencil and mark the point of application of sample by another line 2.5 cm in length parallel to and 5 cm from the mid-line (for the vertical tank) or 7.5 cm away from the mid-line for the horizontal tank.
Now apply the paper in such a way that the two ends of the paper dip in the two buffer compartments adjoining the electrode compartments. Replace the lid of the tank. In about 2 hours-time the whole paper is soaked with the buffer.
Now apply 10 µ1 of the serum (or plasma) by a micropipette on the line marked for sample application. Replace the paper in its place and cover the tank. At a time, several such strips can be set up for different samples. Now connect one electrode with the cathode and the other with anode of direct current with constant voltage.
The electrode nearer the site of application is connected to the cathode. Switch on the current and allow the electrophoresis to run for 16 to 18 hours. A voltage gradient of 3.2 volts per cm length of the paper, or a current of 1.5 to 2.0 milli amps for each 5 cm wide strip is suitable.
At the end of the desired length of time, first disconnect the instrument from the mains, remove the strip from the tank, and dry it at 100°C to 110°C for 30 minutes.
Staining:
Pour sufficient stain in a large size glass or enamel tray and put the dried paper strips in it for at least 6 hours (overnight staining is always preferable). After removing from staining tray, wash for 6 minutes each time in two changes of wash solution, then in the fixative solution for a further 6 minutes. Dry the paper in air or in an oven at 100°C – 110°C.
After staining 5 bands (Fig. 46.5) corresponding to albumin, α1-, α2-, β, and γ-globulins are noticed. It is possible to guess from the electro- phoretogram if some protein fraction is absent, present in decreased or increased amount, or some extra band such as myeloma protein is present.
However, the relative amount of different fractions can be accurately estimated by scanning the paper under photoelectric scanner or different bands are cut and eluted in 6 ml of 0.01 N. NaOH for 30 minutes. These eluted coloured solutions are read in a photoelectric colorimeter at 540 mµ.
Reagents:
1. Buffer (pH 8.6, ionic strength 0.083 M):
Dissolve 3.12 grams of diethyl-barbiturate in water and make up to 1 litre.
2. Dye solution:
Add 25 ml of 95 per cent ethanol to 0.1 gram of bromphenol blue and 50 grams of ZnSO4. Mix well, add 5 per cent acetic acid (V/V), mix to dissolve and make up to a litre with further 5 per cent acetic acid.
3. Wash solution:
Acetic acid (5 per cent, V/ V).
4. Fixation solution:
Acetic acid (5 per cent) containing 0.3 per cent of sodium acetate (CH3COONa, 3H2O).
Gel Electrophoresis of Serum Protein:
(i) Microscopic glass slides coated with gels of agar, starch and polyacryl amide can be used.
(ii) Gel electrophoresis is done in about 30 minutes by which a lot of time is saved.
(iii) Molecular sieving factor helps in better resolution causing in adsorption. Problems and optical measurements are done with better accuracy.
(iv) Different fractions like prealbumin, post albumin, haptoglobins, etc. are separated by starch column electrophoresis.
(v) Many additional fractions of serum proteins are separated by means of disc electrophoresis using polyacrylamide gel. In this disc electrophoresis, the polyacrylamide gel is used as a vertical cylindrical column about 10 cm long and the serum is applied on the top.
The process is conducted with the use of suitable buffers. When the gel is taken on glass plates, it is called an open block type of electrophoresis. Disc electrophoresis is the column zone type, whereas agar slide electrophoresis is the open block type.
(vi) In the immuno-electrophoresis, the proteins of serum are first separated by ordinary electrophoresis on agar slides. Then these proteins are allowed to be reacted upon by specific anti-serum from rabbit, horse, etc. by applying the same in the same agar slide parallel to the path of proteins.
The antibodies of the immune serum diffuse and meet the antigenic proteins resulting in the antigen-antibody reaction with the formation of opaque lenses in the form of precipitins. About 30 fractions of serum proteins have been detected by immuno-electrophoresis.
Isoelectric Focusing:
(i) This is a very recent improved type of electrophoresis which shows the heterogeneity and micro-heterogeneity of proteins and enzymes. Proteins move under the influence of applied electric current as primary gradient in the presence of the secondary gradient pH resulting in the molecular species to resolve further at equilibrium position. The pH of buffer in the usual electrophoresis does not vary.
(ii) Proteins or enzymes are put into a system of suitable carrier ampholytes (e.g., Poly-amino-poly-carboxylic acid) and electrolysis is carried out. The more acid ampholytes with a lower iso-electric point move to the anode and the more alkaline ones with a higher isoelectric point move to the cathode.
Thus the pH shows increasing values from anode to the cathode. Hence each protein or enzyme focuses quickly at its respective iso-electric pH in the resulting pH gradient.
(iii) In this technique, the micro-heterogeneity has been evaluated for gamma globulins, ovalbumin’s, globin chains of hemoglobin, myeloma proteins, lipoproteins, a-fetoproteins, etc. Aldolase is separated into five components and L-amino acid dehydrogenase is separated into 18 components with iso-electric pH values ranging from 5.2 to 8.4.
Enzyme, Enzyme- substrate complex, enzyme-NAD combination, etc. have been shown by this process.
(iv) The immuno-isoelectric focusing involves the coupling of isoelectric focusing with immuno-diffusion in which the micro- heterogeneity of protein antigens can be analysed easily.