Background with chicken primary antibody - (Nov/10/2005 )
I'm having huge background problems on my western blots when using a chicken IgY primary antibody. I get so much background with it that it obscures any bands that appear. I've tested the secondary antibody by itself and it's fine. No background whatsoever so I'm guessing all the background is coming from the primary antibody. Does anyone have suggestions on what to use for blocking to avoid this problem or any protocol changes that would help. Currently I block with TBS+0.1% Tween 20+5% skim milk and wash with TBS+0.1% Tween 20. The detection is done by ECL. Any help would be greatly appreciated. Thanks.
you may increase blocking time (up to 2h RT). If your secondary ab do not get background with others primary ab at same dilutions, it's not dilution of it the problem, but you may have to less dilute the first ab.
What is the exposure time? quite short (less than 30'') or longer?
you can also reduce washing stringency and general by doing a 0.05% tween solutions.
finally, sometimes results are better with PBS instead of TBS.
There is a company that makes a blocking reagent specifically for chicken antibodies for exactly your blocking issues I believe.]
They make a blocking reagent.
never used it..but would love to know how well it works....Let me know if you get it.