EXPERIMENT-2

ROCKET IMMUNOELECTROPHORESIS

By: Abhishek Shetye

Sharvari Sangle
Shital Patil
 Introduction:

 Rocket Immunoelectrophoresis is an adaptation of radial

immunodiffusion developed by Laurell.

 It is called as “Rocket Immunoelectrophoresis” due to

precipitation bands in the shape of cone like structure i.e. rocket
shaped at the end of the reaction.

 The antigen migrates in an electric field in a layer of agarose

containing an appropriate antibody.

 This antigen migrates towards the anode and give rise to rocket
shaped patterns of precipitation. The area of the rocket is
proportional to antigen concentration.
 Principle:

 Rocket Immunoelectrophoresis is a quantitative one-dimensional

single electro-immunodiffusion technique.
 In this method antibody is incorporated in the gel and antigen is
placed in wells cut in the gel.
 Electric current is then passed and the antigen moves out of the
well and enters the agarose gel, it combines with the antibody to
form immune complex which becomes visible.
 During the initial phase there is considerable antigen excess over
antibody and no visible precipitation occurs. However, as the
antigen sample migrates further through the agarose gel, more
antibody molecules are encountered that interact with the antigen
to form immune complex.
 This results in formation of a precipitin line that is conical in
shape, resembling a rocket.
 Applications:

1. Rocket electrophoresis is used mainly for quantitative

estimation of antigen in the serum.
2. The method has been used for quantization of human
serum proteins before automated methods became
available.
3. Determining the concentration of a specific protein in a
protein mixture.
4. In estimation of immunoglobulin protease activity.
5. In enzyme activity electrophoresis.
 Materials:

1. Diphtheria toxoid (Ag) : 3300 If/ml

2. Tetanus toxoid (Ag) : 3600 If/ml
3. Diphtheria antitoxin (Ab) : 300 If equi/ml
4. Tetanus antitoxin (Ab) : 500 If equi/ml
5. IX TAE buffer (pH)
6. 1% Agarose
7. Glycerol
8. Electrophoresis apparatus
9. Well borer
10. Coomassie brilliant blue
11. Destaining solution
 Protocol:
1. Prepare 25ml of 1% Agarose. When the temperature of the
molten gel drops to 55 degree Celsius, add 1.5ml of
Diphtheria/Tetanus antitoxin (antibody) to the molten gel and
mix well. (1ml of antibody per 20ml of agarose gel.)
2. Cast the gel and allow solidifying. Once the agarose gel has
solidified, punch wells using a well borer keeping a distance
of 1cm between the wells.
3. Perform dilutions of Diphtheria/Tetanus toxoid (antigen) to
obtain different concentrations of antigen which is then loaded
into the wells.
4. Load 20ul of the different concentrations of antigen into the
wells along with glycerol to help antigen sink into the well.
5. Electrophorese the loaded gel at 5 volts/cm till precipitation of
antigen-antibody complex i.e. rockets, is observed for the well
containing highest concentration of antigen.
6. Remove the gel from electrophoresis apparatus and measure
the length of the rockets.
7. Plot a graph of height of rockets (in mm) vs concentration of
antigen.
8. Stain the gel for better visualization of the rockets.
 Protocol for staining:
1. Wash the gel in saline (overnight) to remove the excess
antibodies from the gel. Then repeat saline wash twice, this
time keeping gel in saline for 2 hours per wash.
2. Drain the saline and flood the gel with distilled water for 2
hours to remove excess salts from the gel.
3. Wash the gel twice in distilled water after 15 minute
intervals and then drain all the water.
4. Place the gel on a plastic support and dry the gel completely.
5. Once dried, immerse the gel with Coomassie brilliant blue
dye for 2-3 minutes and discard the dye.
6. Immerse the agarose gel in destaining solution and keep
shaking gently which is followed by second destaining.
7. After all the excess dye has been removed the rockets
stained blue will be clearly visible.
 Observations:

A) For Diphtheria toxoid – antitoxin:

Dilution Concentration of Height of Rocket

Antigen(lf/ml) (mm)

Undiluted 3300
1:2 1650
1:4 825
1:8 412.5
1:16 206.25
Unknown
B) For Tetanus toxoid – antitoxin:

Dilution Concentration of Height of Rocket

Antigen(lf/ml) (mm)

Undiluted 3600
1:2 1800
1:4 900
1:8 450
1:16 225
Unknown
Graph :
Result :
 Limitation:
• The antigen has to be negatively charged for
electrophoretic movement within the agar matrix.

• Nor is it possible to measure the amounts of several

antigens in a mixture at the same time.

• Some proteins, immunoglobulins are not sufficiently

charged to be quantitively analyzed by Rocket
Immunoelectrophoresis.
 References:

 Walker JM. Rocket immunoelectrophoresis. Methods Mol

Biol. 1984;1:317-23. doi:10.1385/0-89603-062-8:317. PM
ID: 20512702.
 Kindt. T.J., Osborne B. A. and Goldsby R. A. (2006). Kuby
Immunology, 6th Edition, W.H. Freeman and Company
 https://microbenotes.com/rocket-immunoelectrophoresis/
 https://experiments.springernature.com/articles/10.1007/97
8-1-60327-259-9_137

 https://www.genome.gov/genetics-glossary/Electrophoresi
s
 https://microbenotes.com/rocket-immunoelectrophoresis/#
principle-of-rocket-immunoelectrophoresis