signal is weak or fading - (Feb/07/2008 )

Hi,
I have some new strange problem with my westerns.
Antibodies that earlier have given very good strong signals are now weak, and Ab that are weak are now not giving anything at all. When I develop my membrane I see no signal at all (blank membrane) or I can detect weak signal at very short exposure times but when I try longer exposure the membrane is blank.
I am using the same protocol, reagents, type of cell lysate etc. as before, so I know that it should work for this conditions. The ladder looks ok so transfer should work.
I have read that too much 2nd antibody can give a blank membrane, but since its the same that I have used before I don´t understand why this should happen now.
Does anyone have any suggestions???

Ps. I have already tried to do new solutions.

Is your developer solution still fresh (if you are doing ECL-exposure and x-ray detection) ? If it is old, you get no signal at all at some point and only weak ones the time before.

Are you using to much blocking reagent or too much Tween? Too extensive washing?

Agree to check your developer solution. I had this same problem with the Pierce femto kit. The bottle says that the shelf life is good for six months at room temp. We kept ours in the fridge and at 4 months it started getting weaker.

Thanks for your answers,
but I don´t think thats the problem. I am using an "home made" ECL solution that has worked before - and I have made new ones from scratch and its still not working (But I think I should try again).
Blocking, tween and washing steps are all as I have done before.... so it should work....

Do you suspect any changes in antibodies, primary and secondary?

If you're using a home-made ECL.... is your peroxide fresh? Not just the working solution, but the stock also?

i had a similar problem and i found out that my primary antibody went bad.

Even if your ECL is well, you should have a look on your developer solution in which you put your x-ray films into. If this is deep brown or black, then your films won't develop right and you will not see a signal at all or just very few.
Sure, antibodies can get bad, you have to check how old they are also. I once found that my second antibody has gone bad after too much freezing and thawing, so now I freeze small aliquots. You can test your ECL with a bit of second antibody to see if it is glowing in the dark. If this works fine, than it will be something other.

Hmmm, I might have found out why everything stops working after a while. It looks lika the stock solution of PBS crystallize after a couple of weeks. Maybe this can block the membrane? It sounds a bit strange, but after I have started to filtrate the PBS it works.
Does anyone belive it could be so?