Originally Posted by mauer
Thankx for your response and the link to the document. Platers were sealed, it's just that we usaully do overnight coating at 4 degrees and I was concenred if diffrences in results will be attributed to difference in coating temperature. (perhaps one can always argue that, but i just wanted to double check that i am not processingplates that shouldn't be processed)
Coating differences in regard to temperature affect you if:
1) Antibody is not stable at room temp, i.e. it degrades. You can check this out on the product info. Sera (sometimes sold as Ab, always labeled sera, though) may pose this problem, purified antibody usually does not.
2) If pH is not stable, overnight at room temp. pH does affect binding, you should be fine with pH > 8, but acidic pH would remove bound antibodies.
Most likely you are fine because:
1) Surface coating is determined by surface chemistry and usually results in a geometric spacing of the antibody (or protein) across the polypropylene well. (the greater the conc of Ab the closer the spacing, the less Ab the greater the spacing; you see a pattern either way).
2) Either way RT overnight or 4C overnight--you are probably binding the max amount of Ab. Therefore less likely to see a difference due to greater/lesser capture Ab on your wells.