Western Blotting - Antibodies

1. Load gel in apparatus as described by manufacturer.

2. Transfer proteins according to above ensuring that the NCM is toward the positive electrode. When using the Hoefer apparatus be sure to either precool the buffer and run the unit in a cold room or cool the unit with a refrigerated circulating bath.

3. Immerse the nitrocellulose sheet in chosen blocking solution and incubate for 60 minutes to overnight at RT on a rocking platform.

4. Incubate the sheet in antisera for 90 minutes. Normally a 1:100 dilution is sufficient for this procedure. The antisera may be diluted in blocking solution for higher stringency or in TSN. In order to minimize the amount of antisera, place the sheet in a 150mm diameter petri dish, or in containers for the hybridization chamber. One can also cut the sheet into strips, etc.

5. Wash the sheet in TS for 10 minutes, twice in TSN for 20 minutes each, and then again in TS for 10 minutes. When higher stringency is required, wash blot 4 times 15-20 minutes in blocking solution.

For Iodinated antibodies or Protein A:

6. Incubate the sheet with the Iodinated antibody or Protein A diltued in TSN (100,000 cpms/ml) for 30 minutes.

7. Wash again as in #5-all wash solutions will be radioactive so the appropriate care must be taken!

8. Air dry and autoradiograph.

For enzyme labelled conjugates:

6. Incubate the sheet with appropriate conjugate diluted in blocking solution. Normally a 1:1000 dilution is sufficient.

7. Wash once 15 minutes with blocking solution, twice with TS for 15 minutes, once with TSN for 15-20 minutes and finally once with TS for 15 minutes.

8. Develop color using the appropriate substrate: For HRP conjugates, mix 5 parts TMB peroxidase substrate, 5 parts TMB solution B, and 1 part TMB membrane enhance. For AP conjugates make the following solution: 0.03g Napthol AS-MX Phosphate, 0.06g Fast Red in 40 ml of 2g/800 ml Tris, pH7.5. Stop reactions by rinsing with water.