Western Blot no signal - (Aug/16/2011 )

Hi, im not getting a signal from my western blot doing exactly the same blot ive done a number of times before with exactly the same reagents except new PBS-T. This happened now three days in a row with 6 different proteins. Coomassie and silver stained SDS-PAGE looks good, transfer is ok (marker is transferred). What happens is after I incubate the membrane with the supersignal femto or pico (2 different batches of each tried) for 1-5 minutes the signal dies in about 5 minutes as developed using 2 different machines or in 3 fresh cassettes with 2 different sources of film. I checked the pH of the PBST and it was around 7.2 which I guess should be ok. The problems really started after we changed the 10xPBS used to make the PBST and for some reason although I keep trying to make new buffer it doesnt change the result (I tried now 3 buffers). Anyone have any ideas or similar experience?

sounds like you have too much secondary antibody. you are using up the substrate too fast. reduce the protein load or dilute the antibody solutions.

if your background is too high then replace your block and/or wash longer and/or add more tween to your pbst.

I tested antibodies: 10 fold dilutions of primary and secondary had no effect, i.e. still see a signal after 2 minutes but dies off after 5 or so.

are you seeing a lot of background or just very strong bands?

if background then block or wash better.

if bands then you may need to dilute the antibodies more (are you using a new lot of antibody? it may be higher titer).

Yeah I see no bands. Same antibody (and tested others) as I always have used before.

Maybe the HRP on your antibody is starting to go? Have you tried a different secondary antibody or just different primary antibodies with the same secondary?

5 different secondary antibodies with different proteins (in the most important experiment 2 different secondary antibodies)