Primary Ab non-specific binding - How to check for it? (Nov/25/2009 )
Hi,
I am wondering how can I properly check for non-specific binding of primary (unlabeled) antibody? I can imagine how to do it for secondary Ab (use directly sec. Ab in sample w/o primary ones) but for primary Ab...? Am I just blind not seing trivial solution? 
Thank you for any suggestions,
Paja
Western using the primary - if you get one band (of the right size), then the Ab is specific.
or if u don wanna waste time in running a gel.. take a positive and a negative (any related or unrelated protein family) control and slot blot it and develop using primary and secondary and see... tat will save u the running time and u can know the specificity too!!!
Also you can decide the titre of your primary cause many slots can be blotted in teh apparatus!!
Good luck!!
Paja on Nov 25 2009, 12:31 PM said:
I am wondering how can I properly check for non-specific binding of primary (unlabeled) antibody? I can imagine how to do it for secondary Ab (use directly sec. Ab in sample w/o primary ones) but for primary Ab...? Am I just blind not seing trivial solution?
Thank you for any suggestions,
Paja
I agree that western blotting can help in determining antibody specificity. However, many abs have conformationally-dependent epitopes. Immunoprecipitation followed by PAGE is probably a better option.
klinmed on Nov 26 2009, 03:33 AM said:
Paja on Nov 25 2009, 12:31 PM said:
I am wondering how can I properly check for non-specific binding of primary (unlabeled) antibody? I can imagine how to do it for secondary Ab (use directly sec. Ab in sample w/o primary ones) but for primary Ab...? Am I just blind not seing trivial solution?
Thank you for any suggestions,
Paja
I agree that western blotting can help in determining antibody specificity. However, many abs have conformationally-dependent epitopes. Immunoprecipitation followed by PAGE is probably a better option.
Good idea!