lost signal - (Jun/12/2009 )

Dear,
after weeks of optimizing my westerns (lowering background, enhancing signal...) I finally got the result/protocol i wanted. My boss told me to repeat it one more time and since then........no signal anymore! I changed nothing, but what worries me: went i put my film on the blot (HRP-ECL detection with films, we are saving for a scanning device) after 1 minute i have an acceptable result, when i put a second film on the blot and wait for 5 minutes..you would expect a 5times brighter signal (we use ECL Plus) right?! Nope, my signal is much much fainter! I don't get it, i tried secondary antibody and ECL from collegaes, no difference. I told a friend about it, and their lab had had the same problem, they refreshed everything, no result...untill one day everything worked again normal out of the blue. Anyone experience AND found a solution?
Many Thanks (getting really desperate)

I think you should mix the ingredients of the detection reagent immediately just before detection step and no need to bring there temperature to room temperasture). Also try to use Millipore enhanced ECL kit

Hello,

I also have the same problem? Everything worked so fine and now cat get anything out. I have made new buffers and ordered everything except primary antibody again..but nothing. Have you found a solution yet?

maris on Jul 8 2009, 04:37 PM said:

Hi,
Everything is working fine again...i did order fresh primary Abs (though i tried some frozen ones on my blots and they did work fine, so not sure if that's the reason) and i suspected the water i'm using for diluting the buffers (came out of barrels standing there for quite some time), so i used water right from the tap...and now i have bright bands again.
I also think that if you're trying too hard to get a protocol back working you better let it rest for a while...sometimes your 'mojo' will come back

Actually i was facing the same problem. When i put my film on the blot and i developed it after 1 minute i have an acceptable result. But when i put a second film on the blot and wait for 5 minutes, 30 min or 1h ..I expect a brighter signal (we use ECL west pico from Pierce) !! but it's not the case, sometimes i don't see anything !!! some of my freinds advice me to mix immediately in the dark room since one of the reagent is very sensitive to light !!! i tried to do it with the same kit from another lab, i have obtained good signal after 5 min, but again when i kept the film for 30 min, the signal disapear completly !! any suggestions please ??

thank you by advance,
Amanda