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Methodology for Amino Acids and Proteins

 
     
 
Rocket Immunoelectrophoresis
 
A number of methods are available for the quantitative estimation of a particular protein in a mixture. These include electrophoresis and immunochemical methods. The rocket immunoelectrophoresis is such a simple, rapid and reliable method, since rocket-like immunoprecipitate is formed when the desired protein (antigen) is electrophoresed in an agarose gel containing its monospecific antiserum. Since the height of the peak depends on the relative excess of antigen over antibody, a comparison of the peak heights of the unknown and standard samples allows the unknown protein concentration to be determined. This method was first described by Laurell in 1966. Although it is widely used in the clinical laboratories, quantitative estimation of seed proteins etc., could be easily carried out.
 
 


Principle
When the protein sample (antigen) is electrophoresed into the agarose gel containing the monospecific antiserum in the presence of excess antigen, the Ag-Ab complex is soluble but as the antigen advances, more antigen molecules combine with antibody until and equilibrium is reached. At this stage, the Ag-Ab complex is insoluble, resulting in the precipitation spreading as a rocket from the antigen well. The area of the height of the rocket is then a measure of antigen concentration.
 
 
Materials
0.06M Barbitone Buffer             (pH 8.4)
Sodium Barbitone                       10.3g
Barbitone                                      1.84g
Water                                             1L
Electrode Buffer is prepared by diluting it 1:1 with distilled water
2% Agarose (electrophoresis grade) in distilled water
Antiserum to the protein under investigation.
Staining Solution: 0.1% Coomassie brilliant blue R250 in 50% methanol & 10% acetic acid
Destaining Solution: 10% Methanol 7% acetic acid
Glass plates, size 5 x 5 x 0.1cm or 7.5 x 7.5 x 0.1cm
Electrophoresis System
 


Procedure
1.
Place a clean dry glass plate (5 x 5 x 0.1cm) on a leveled surface.
2.
Melt the 2% agarose either in a boiling water bath or in an autoclave. Transfer the flask to a water bath at 52°C. stand a 5mL graduated, wide mouth pipette in this solution. Allow some minutes to cool the agarose solution in 52°C, and at the same time warm up the pipette.
3.
Place a test tube in a water bath and add 2.8mL of 0.06M barbitone buffer to the test tube. Leave this for 3-5min to warm up. Then quickly transfer 2.8mL of agarose solution to this tube using the wide mouth pipette. Otherwise agarose will set in the pipette. Briefly mix the contents of the tube and return to the water bath.
4.
Add 25-50mL of antiserum to the diluted agarose solution and briefly mix for even dispersion of the antiserum. Return to the water bath to allow any bubbles to settle out. (The appropriate volume of antiserum to be used depends on the antibody titre and has to be determined by trial and error).
5.
Pour the contents of the tube slowly onto the glass plate keeping the neck of the tube close to the tube. Surface tension will prevent the liquid running off the edge of the plate. If necessary, tape can be used to form an edging to the plate. The gel will be approximately 2mm thick.
6.
Allow the gel to set for 5-10min and then make holes using a Pasteur pipette attached to a water-pump vacuum at 0.5cm spacings 1cm in from one (cathode) side of the plate.
7.
Place the gel plate on the cooling plate of the electrophoresis tank. Pour the electrode buffer into the tank compartments. Place the electrode into the tank compartments. Place the electrode wicks (five layers of Whatman No.1 prewetted in electrode buffer) over the edges of the gel. The wicks should not overlap the sample wells. The wells should be at the cathode end.
8.
Deliver suitable aliquots (1-5mL) of sample of each well using a microsyringe. The samples should include a few different concentrations of the pure protein (antigen) and a few dilutions of each unknown sample. Loading of the wells should be completed at the shortest duration or a small current (1-2mA) may be applied during loading in order to avoid any diffusion of the sample.
9.
Immediately after loading, raise the current to 20mA and run for 2.5-3h. Gels can also be run at 2-3mA overnight. In any case, efficient cooling of the plate is essential.
10.
After the run, remove the gel plate from the apparatus. Precipitation rockets can be seen in the gel.
11.
Prior to staining, wash the gel with several changes in saline solution for 4-5 hours to completely remove the unreacted proteins. The gel may be stained wet or after drying to give a permanent record.
12.
The gel is dried by blotting. With the gel on a sheet of clean glass, place 6-10 sheets of Whatman No. 1 filter paper on top of the gel and apply heavy weight for 2-3h. Then the top wet filter papers are carefully
removed to reveal the flattened and nearly dry gel. Further drying of the gel is continued with a stream of warm air using a hair-dryer.

13.
The wet or dried gel is now gently shaken in the staining solution for 15-20 min and subsequently destained.
14.
Measure the height (mm) of each rocket and plot a graph of peak height versus concentrations of standard.
15.
The protein concentration in an unknown sample is calculated from the graph.
 
 


Notes
1. Small aliquots of 2% agarose solution can be stored in separate tubes and each time a tube withdrawn fresh for use.
2. It is essential to standardize the dilution of the antiserum and the concentration of the standard and sample proteins by trial and error runs for the right size of the rockets for comparison.
 
 


References
1. Laurell, C B (1966) Anal Biochem 15.
2. Walker, J M (1984) In: Methods in Molecular Biology Humana Press New Jersey Vol. 1 pp 317.

 
     
 
 
     




     
 
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