1.5. Rocket immunoelectrophoresis
Rocket immunoelectrophoresis is a simple, quick and reproducible method for determining the concentration of a single protein in a protein mixture. Like immunodetection following western blotting it is a method based on the affinity of a specific antiserum (which can be mono- or polyclonal) with a specific protein. As it does not use a secondary antibody conjugated with a moiety for high sensitivity detection, but is based on the formation of an antigen-antibody precipitate in a gel matrix. It does not have the sensitivity of the immunodetection method described in section 1.4. It does, however, have the advantages that the presence of a specific protein can be analysed fairly quickly, both qualitatively and quantitatively, in a relatively large number of samples.
In this procedure, appropriately diluted samples are applied to small circular wells cut into an agarose gel which has a specific antibody already incorporated in its matrix. When migrating in an electric field, the protein of interest will eventually reach a critical point of antigen-antibody concentrations resulting in the local formation large precipitating complexes. The agarose gel can then be stained and the rocket-shaped precipitate becomes apparent (Fig. 4). The position of this peak is directly related to the concentration of the protein of interest.
A typical rocket immunoelectrophoresis assay of honey bee haemolymph proteins is done as follows:
1. Prepare a 1 % (w/v) agarose solution in 0.06 M Tris-HCl buffer, pH 8.6.
2. Completely dissolve the agarose by boiling for 2-3 min.
3. When the agarose has dissolved, place the flask in a 52 ºC water bath.
4. Once the agarose solution has cooled to 52oC (use a thermometer to check temperature), add an appropriate amount of antiserum (this amount has to be determined empirically by serial dilution assays for each antiserum).
Do not add the antiserum earlier as a higher temperature will cause its denaturation, also do not add it much later, as agarose will soon start to solidify; briefly mix to ensure even dispersal of the antiserum.
5. Pour the agarose solution (15 ml) onto a clean glass plate (usually 10x10 cm) so as to guarantee an even gel thickness (the final gel will be approximately 1.5 mm thick):
5.1. Place the glass plate on a levelled surface.
5.2. Start pouring the agarose solution in the middle of the plate.
The liquid will spread to the edges of the glass plate, but surface tension will prevent it from running off the edge of the plate (alternatively, tape can be used to seal the edges).
6. Wait for 5–10 min for the agarose gel to harden.
7. Using a steel puncher connected to a suction device punch holes of 1 mm diameter at minimal spacing of 0.5 cm forming a line across the plate at approximately 1 cm from one edge.
The holes can also be made using a glass Pasteur pipette with a suction bulb.
8. Place the glass plate with the gel on a horizontal electrophoresis system.
9. Fill the troughs with electrode buffer (0.3 M Tris-HCl buffer, pH 8.6).
10. Apply wicks (made from filter paper) immersed in buffer to form a bridge between the gel edges and the buffer troughs.
Make sure that the gel is in correct orientation with respect to polarity of the electrophoresis system [the cathode (-) should be next to the sample wells].
11. Add gel buffer (0.06 M Tris-HCl buffer, pH 8.6) at a 1:1 ratio (v/v) to the appropriately diluted samples.
It is important to ensure that all samples are prepared to an approximately equal volume and, if possible, contain similar amounts of total protein.
12. Apply samples to the well.
This should be done as quickly as possible to minimize diffusion from the wells into the gel (alternatively a small current of 1-2 mA may be applied during loading to overcome diffusion problems).
13. Immunoelectrophoresis is then carried out at 20ºC for 16 h, at a setting of 0.08 V/cm gel length (make sure to check polarity).
14. For staining after electrophoresis:
14.1. First, cover the gel with two layers of filter
paper soaked in saline 0.9 %.
14.2. Add a 2–3 cm thick layer of dry soft paper tissue.
14.3. Cover with a thick glass plate to guarantee application of slight and even pressure (about 10 g/cm2).
15. After 20-30 min the gel layer should have now been reduced to a thin film covering the glass plate.
16. Immerse the gel for 24 h in saline solution.
17. Wash in distilled water for 30 min.
18. Cover the gel with two layers of filter paper soaked in distilled water.
19. Cover with a 2–3 cm thick layer of dry soft tissue paper.
20. Cover with a thick glass plate to apply slight and even pressure (about 10 g/cm2).
21. Air dry the gel on the glass plate with a hair dryer.
Hot or cold air can be used.
22. Place gel for 20-30 min in staining solution made up with 0.25% (w/v) Coomassie Brilliant Blue R-250 dissolved in a solution 5:5:1 (v/v) of ethanol, ddH2O and glacial acetic acid (see section 1.3.3).
23. Remove excess dye by washing the gel in the same solution prepared without the dye.
24. After drying at room temperature the gel can be kept as a permanent record.
25. Measure the peak height of the rocket-like precipitates for each sample.
quantification compare this to a standard sample for the protein of interest
run on the same gel; for relative quantification set the sample with the
highest peak as 100 %.
Fig. 4. Rocket immunoeletrophoresis for quantification of haemolymph vitellogenin in 1 to 6 day-old Apis mellifera workers reared on different diets. Haemolymph was from bees fed a 0 % pollen diet (wells 1-4), a 15 % pollen diet (wells 5-7), a 50 % pollen diet (wells 8-10), a pollen-free sugar diet supplemented with soybean and yeast (wells 11-13), and naturally fed workers (wells 14-16). Reproduced from Bitondi and Simões (1996). Copyright Journal of Apicultural Research.