After running the acrylamide gel, transfer it to nitrocellulose by electrophoresis. The gel is put in the transfer apparatus with the nitrocellulose on top of it, facing the positive electrode. The transfer should be done in the transfer solution, at 4°C, 20 volts, overnight, or at 50 volts for 4-5 hours.
After the transfer, folow the procedure below:
1) If you want to check the transfer you could stain the filter with Ponceau S stain
(reversible):
(a) Stain for 5 min.
(b) Destain in water for 2 min.
(c) Wash off the stain in PBS for 10-30 min.
2) Place nitrocellulose in a Seal-a-Meal bag with blocking solution. Incubate and shake for at least one hour at room temperature (can be left overnight).
3) Rinse briefly in TTBS.
4) Incubate with primary antibody diluted in blocking solution for 1-2 hours at room temperature (shaking). Proper amount of antibody is determined empirically. Supernatants from cells, no dilution - 1:5; Ascites or serum - 1:100 - 1:500.
5) Wash twice for 10 minutes in TTBS.
6) Incubate with secondary antibody diluted in blocking solution at room temperature for 1 hr. For AP; or HRP; conjugates, dilute 1:500 - 1:1000. For 125I-labelled protein A or anti-IgG use 2 x 105cpm/ml.
7) Wash as in step 5.
8) (a) If using 125I, expose nitrocellulose using Kodak XAR5 film and intensifying screen
overnight at -70°C.
(b) If using AP conjugate;, rinse once more for 5 minutes with TBS (no Tween).
Develop blot using the BCIP + NBT substrate system (Bio-Rad kit).