Questions about phospho-protein level in western blotting - (Oct/04/2006 )

Hi all,

I have some questions about western blotting. I tried phospho-protein antibody. I do not why every time there is a lot of background. Anyone please give me some suggestions to reduce the background?

ps: I used ECL substrate.

thanks

-stormlina-

I got less background by diluting the secondary AB in 3% powder milk in TBS1x instead of TBS1x alone. Hope this helps a bit.

-pumuki-

if you're blotting phospho-proteins, you may want to switch away from milk as a blocking agent. some people say it makes no difference, but many people think it does...and I can tell you from my experience that it reduced background in my phospho-westerns when I switched to 3% BSA/in TBS-tween

-aimikins-

sometimes it also depends on Ab itself; we used lots of different anti-phospho Ab where we have often minor background with monoclonals; we use non-protein commercial blocker (rotibloc)

-The Bearer-

QUOTE (kosmodrom @ Oct 4 2006, 05:22 PM)

sometimes it also depends on Ab itself; we used lots of different anti-phospho Ab where we have often minor background with monoclonals; we use non-protein commercial blocker (rotibloc)

you may want to try blocking with bsa instead of milk, it definately reduced the background for me at least.

-Jimmy_september-

Thanks all for the nice suggestions. One more question, anyone has used pAkt (Thr308) antibody from Upstate? If so, how does it work?
Thanks.

-stormlina-