Questions about phospho-protein level in western blotting - (Oct/04/2006 )
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.
I got less background by diluting the secondary AB in 3% powder milk in TBS1x instead of TBS1x alone. Hope this helps a bit.
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
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.
Thanks all for the nice suggestions. One more question, anyone has used pAkt (Thr308) antibody from Upstate? If so, how does it work?