About the probing the Streptavidin-HRP - western blot (Sep/29/2006 )
Acutally i use 1% milk powder + wash buffer (TTBS) probing the membrane, i want to ask
Is it better to use higher % of milk powder (up to 5%) during the probing?
Also, i want to know how can I reduce the backgroup significantly (Streptavidin-HRP)?
I don't think it's better or worse to use any percentage of milk, other than it's a function of how sticky the antibody is. I usually use 2%, but will go up to 5% only if I need to decrease background. For me, this works. You can't use avidin-biotin systems with milk because milk has endogenous biotin (I think that's why), so this will cause high background. You should use BSA instead.
Even though there are endogenous biotin in the milk, but it does not affect the result, right?
It won't interfere with the chemistry, no. If your detection and imaging methods are sensitive enough to pick up the signal over the background, then you should still see bands. I'm just worried that your background will be so high that you won't be able to visualize your bands as easily as without the milk.
Sorry, i also want to know if the streptavidin-HRP concentation is too high, then does it produce the high-background result?
It shouldn't produce background if you use BSA as your blocker. That is why people use avidin-biotin systems, to lower background and increase signal. If you are using the milk, though, I would imagine that the higher the streptavidin-HRP concentration, the more background because there would be more interference with the milk proteins. But I have never tried that.
I reduce a lot of background if I dilute secondary Ab (HRP) in milk powder 3% in TBS1x instead of only in TBS1x.