Western blot film's stained black all over?

[missyJ]

hi all,

Every time I develop a western blot film it's stained black everywhere that I can't see any band at all. After adding the pri antibody, i wash the membrane 5X and then add the sec antibody and wash again. Then i'll develop the film using the some substrate. The thing is, after adding the substrate, my membrane develops the bands that I'm interested in obtaining, but when I develop it on a film the whole film's stained black such that the bands cannot be observed. Anyone else has this prob and any recommendations?

[admin]

Hi
Did the membrane glow in the dark when exposing? How much secondary antibody did you use / dilution ratio used?

[missyJ]

yes the membrane did glow in the dark. I used only 0.5microliters of sec antibody...10000X dilution! but i remember most parts of the membrane were glowing, not just the bands. does that mean the blocking/washing wasn't enough? I blocked for 1hr using 5% non fat milk in PBS+Tween before adding the primary antibody...

[admin]

Yeah,
although cool that is always a secondary issue - maybe you need to run a gel transfer then cut up membrane along lanes - try different secondary dilutions to see what doesn't glow - leads to good bands.
Are you sure you diluted it properly - may have been human error?

You may have to dilute your secondary more, or best to wait a while to let your membrane "cool down" ie not glow - maybe will take 30 min - 1 or 2 hours lol then do a quick exposure ie 2-5 seconds. How long did you originally expose for?

[missyJ]

yea i'm quite sure i diluted correctly because the first time i did i used 2microliters of sec antibody and the same thing happened, so this time i used only 0.5 microliters. I exposed it only for a while - a few seconds and it already turned all black. it's weird cos' my membrane itself is stained with the 4 bands upon addition of the substrate but the film, when developed, is stained fully black. but yes, the whole membrane does glow in the dark...!

[manders77]

Maybe your block is inappropriate for your antibodies? Try a different blocking solution, maybe BSA or gelatin? And sometimes secondary antibodies need to be diluted A LOT more than 1:10,000 - sometimes up to 1:100,000 or more. I also wash my membranes with an excessive amount of wash buffer (almost a liter per blot!), but I never have this problem. For your washing steps, put your membrane in a bigger container and use a large volume of wash with a strong rotating motion. Good luck!