SDS and/or NaOH's affect on ELISA results - (Dec/16/2011 )

Hi Bioforum community,

I would certainly appreciate your input, be it literature supported fact or opinion, regarding my problem. As a bit of background, I am in a field that is a blend between polymer science and biochemistry so I often run into very unusual experiments such as the following: I have to "melt" my polymer/protein matrix in one of the following mixtures; .1N NaOH and 5% SDS, 13.5N NaOH, or almost any water miscible organic solvent (that doesn't precipitate proteins). In order to quantify the minute amount of protein I need to use an ELISA assay (no a simple mBCA assay will not do the trick). My question to you is: will any of these systems not destroy any hope of reliable results from my experiment? If not, does anyone have suggestions?

Many thanks,
Cheers,
Feldman

The assays simply will not work under these conditions. So, the options are to extract the 'melted' or solubilized proteins by size or precipitation and then resolubilize in a compatible solution, or dilute into conditions where the components of the assay will function. Antibody antigen interactions will tolerate salt concentrations, SDS and pH variations to a point but the actual tolerance will be dependent on the individual antibody/antigen. 0.5-1 M NaCl, 0.2% SDS and pH 3 to 10 are all conditions that some antibody antigen binding interactions will tolerate. So a 25 x dilution of your NaOH SDS solution into a neutralizing solution getting the pH in the 4 to 10 range may work, or neutralization of your 13.5 N NaOH with HCl and dilution so that the salt is <1 M may be useful.

Can you 'melt' the polymer/protein and then separate the protein from the polymer prior to analysis?