1 reply to this topic

[feldman]

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

[Ben Lomond]

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?