Fluorescence detection of Western blots - (May/14/2007 )

Hi,

I am going to detect the Western blots by fluorescence for the first time. I have ordered the secondary antibody from Alexa (Alexa Fluor350 goat antimouse IgG). I was wondering if either the blockage step or the incubation of the membrane (nitrocellulose or PVDF) with this secondary Ab must be carried out in 5%-non-fat milk-TBST buffer....or it could be better to use BSA buffer?. Please I need your advice!!!!

Thanks!
Rosa2007

In general I've always used milk for blocking (ECL and fluorescence). On a related note, when I first started doing FL detection I noticed that the PVDF membrane I used (Immobilon-P) gave high background, so I had to switch to Immobilon-FL (low fluorescence PVDF) for more easy detection of some of my not-so-abundant proteins. Also, you may want to add a few extra washes after your secondary, as the Alexa Fluor secondaries, at least in my hands, seem to hang around a bit longer than biotinylated secondaries.

For fluorescent Westerns, try Seablock from Pierce. I don't know why, but it gives less fluorescence background so you will have more sensitivity and better S/N ratio.

http://www.piercenet.com/Products/Browse.c...;WT.mc_id=forum