How much of antibody to add? - (Feb/11/2008 )

Hi all,

I am sure that this question is quite dumb.. But please its been haunting me for a while and not sure how to go about doing it... I am doing western blotting for the first time and for the primary antibody dilutions my boss told me to make a 1 in 500 and 1 in 10,000 dilutions. So i added 10ul in 5ml of 1%milk solution and 1ul in 10mls of 1% milk solution respectively. I would like to know if I need to add the entire 5ml and 10ml into respective membranes or just added 2ml(say from 5ml one) and 5ml (from 10ml one).. Is it ok if I just dunk in the whole 5ml and 10ml into the respective dishes???

I would also like to know how to go about doing the Biotinylated marker.. After transfer in PVDF, I have blocked the membrane with 5% milk solution. Shud I then cut just the marker lane and block with avidin and biotin then ABC reagent and then the ptn samples with primary antibody in 1% milk solution. PLease guide me on that. Thanks. ASlo if anyone has a Westren Bloitting for dummies protocol, please share it, it wud be a great help. Thanks!!!

If my blot required 5ml of solution, then I would make up my antibody in 5ml milk and use them all up.

Add enough solution to cover the membrane.

Thanks a lot!!!!!