exposure times? - (Sep/17/2008 )

hello,

i am trying doing a western blot to detect gfp protein. im having issues when it comes to get autoradiographs. how long do i need to expose my films for? i have tried 1, 5, 10 minutes and have not seen any bands. any help?

The answer is... as long as you need to. As this isn't very helpful, you will need to tell us what detection system you are using and the type of film. as well as how much protein loaded.

Basically the rule is that you can expose until your membrane shows up as black on the film (i.e. background). I have found that for both HRP and Alkaline phosphatase conjugated secondaries this will be about 2 hours exposure.

Note if you are looking for GFP autoflourescence then it isn't going to work, you will still need a primary against GFP and a conjugated/labelled secondary antibody.