After stripping, did you test the efficacy of the stripping process by incubating in secondary antibody and then imaging the blot? And, do you mean both primary antibodies are of rabbit origin?

As diana said, you should check your stripping process. If not try with an old-school stripping technique, which y generaly prefer

.Thanks guys. No I didnt check with the secondary as suggested by Diana. May be I should do it. Nicolas what is the old school stripping and how is it done.

It is very simple. To prepare 100 ml of the stripping solution, add 20 ml of SDS 10%, 12.5 ml Tris HCl

pH 6.8 0.5M to 67.5 ml of water and 0.8 ml ß-mercaptoethanol.

Warm the buffer to 50°C and place the membrane in the buffer for 30-40 minutes with

agitation.

Wash with water several times and with tbs tween. Remwmber that this procedure

removes the blocking from the membrane, so you will have to block again before using the other antibody.

This solution can be stored at 4ºC and used a few times.

Great answers above! I might add that it would be wiser to use the second primary antibody from a different species and to make sure that the secondary antibody to this second primary antibody does not cross-react to the first primary antibody. This is important because the two proteins you like to detect separately are of similar molecular weight.

That is the problem. I dont have the primary and secondary with different species. May be I will try with the home made stripping buffer.