No signal at all - (Jan/25/2010 )
in addition, you prepare the antibody in tbs-t, why do you wash the gel with tbs (after washing with tbs-t) prior to adding the antibody solution? i would just stay with the tbs-t.
and...
you may want to try more concentrated antibodies. you went to 1:5000 with your monoclonal but to 1:2000 with your anti-his monoclonal, try 1:1000, 1:500... until you get a response, especially if your antibodies are old (as clare points out).
have you tried new antibodies?
The antibody is about two years or more. It is stored in -20 C.
And I have to tell you that.. when my friend did the same protocol today using two kinds of substrate (AEC and ECL), using the two-year-old antibody, it worked for AEC and didn't for ECL (nothing on X-ray film).
So, we suggest there's something wrong with either the X-ray film (we use Kodak X-ray film), or the developer/fixer, or the developing process.
We have tried a suggestion from this forum to bring the film outside to the light and develop it, and it was totally black. So, what's wrong?
For your additional info, my friend used 1:5000 for the secondary antibody dilution. Maybe I should increase my antibodies concentration. Thanks for the suggestions! 
About the TBS and TBS-T, maybe next time I can try to stay with TBS-T. Thanks again..
Appreciate your suggestions very much..!
Hmm..so it worked for AEC but not ECL?
Is there anything in the antibody that could inhibit the ECL reaction? My memory is a bit vague here but I do remember that sodium azide in an antibody solution inhibited my detection during IHC.....
Your film should be black if you exposed it to light and then developed it.
Clare
v413n_n on Feb 4 2010, 10:03 AM said:
And I have to tell you that.. when my friend did the same protocol today using two kinds of substrate (AEC and ECL), using the two-year-old antibody, it worked for AEC and didn't for ECL (nothing on X-ray film).
So, we suggest there's something wrong with either the X-ray film (we use Kodak X-ray film), or the developer/fixer, or the developing process.
We have tried a suggestion from this forum to bring the film outside to the light and develop it, and it was totally black. So, what's wrong?
For your additional info, my friend used 1:5000 for the secondary antibody dilution. Maybe I should increase my antibodies concentration. Thanks for the suggestions!
About the TBS and TBS-T, maybe next time I can try to stay with TBS-T. Thanks again..
Appreciate your suggestions very much..!
Yes, sodium azide can inhibit ECL reaction.
Both of my primary antibodies are supplied in a liquid containing 0.1% sodium azide as preservative. However, one of them worked well with ECL before.
v413n_n on Feb 8 2010, 11:23 AM said:
Both of my primary antibodies are supplied in a liquid containing 0.1% sodium azide as preservative. However, one of them worked well with ECL before.
Hmm..well I guess just try the antibodies at lower concentrations as mdfenko suggested?
C
Yes, maybe that will be a good idea...
Thanks a lot!!
We had a problem once in the lab with the ECL and at the end was that somebody mixed both up!! so, just try with a new kit of ECL, maybe it is the problem.
Regarding your protocol:
I would not wash the membrane after the blockin step (directly from the milk to the primary antibody) and I would skip the washes with TBS.
I also think that the antibodie dilutions you are using are too diluted (1:120000??)