western questions - (Dec/07/2006 )
I have two questions about western
1. I found the protein marker didn’t run straight in a higher like 18% gel, but run straight in lower like 7.5% gel. Would someone tell me the reason?
2. There is no problem to probe with primary antibody overnight. Is it harmful to block or put in secondary antibody for a long time? What would be the side effects?
Thanks.
I don't know about question #1, but overblocking could theoretically reduce your signal, while overly long incubations with secondary can raise your background.
Incidentally, there are times when overnight incubation with primary antibody is not a good idea. I've found that some rabbit polyclonal primary antibodies are really dirty and give high background. This can be corrected by reducing the incubation to 1 hour at room temp.
1. I found the protein marker didn’t run straight in a higher like 18% gel, but run straight in lower like 7.5% gel. Would someone tell me the reason?
2. There is no problem to probe with primary antibody overnight. Is it harmful to block or put in secondary antibody for a long time? What would be the side effects?
Thanks.
I block overnight always for my Westerns. For your marker not running straight, how fast were you running it? Usually taking down the voltage (just under 100V) keeps things straight for me, although I run 12% normally. Higher than about 150V and my gel starts to "smile."
If u block overnight so u will have to wash ur memebrane with TBST washing buffer but usually i block for one hour with no wash.
for the first antibody its good to incubate overnight but sometimes it can cause high background according to the type of ur antibody.
for the second antibody i used to incubate for one hour/4 hours/6 hours and for me its the longer the better .