reduction of non-specific binding - (Sep/27/2006 )

Hello!
I have a little problem. I am trying to make a new immunodetecting column. I have problems with non-specific binding. I would like to know which additives should I use to lower the non-specific binding of antibody to my column and that at the same time does not affect the specificity and activity of the antiboies.
Any idea will be sincerely appreciated!

What chemistry are you using to conjugate your antigen or antibody to your column?

Hi!
I bind my antibodies through the amino group to an aldehyde support. Later on, I reduce the aldehyde group to hydroxyl to prevent further binding.

It sounds like you aren't binding enough peptide so that you have extra aldehyde groups that are not being reduced by your subsequent reaction. Can you tweak the reduction reaction in order to catalyze more aldehyde groups? Can you bind more peptide so that there are less available aldehyde groups for reducing? I use the Pierce kit, and they use a pH 10 buffer to couple the peptide to the support that they say helps increase the conjugation density. I don't know that much about the chemistry though.

Hi again!

I make the reduction due to the fact that there is always a possibility left that some of the binding groups on my support remain free (the possibility always exist).
The chemistry though is very simple. First of all I have diol group on my support, which I turn in aldehyde group. Then I use cyanoborohydride to bind my primary antibodies to the column. Some Spanish researcher did the same, so I just copycat them.
The first column I have made was good, then I stared to optimised the method (lowering as much as possible the non-specific binding of the second antibody).

I know it sounds complicated, but … well we will see how is going to end my research.

Thanks a lot for your suggestions.