I have been attempting to calculate the mol/mol ratio of free thiol to protein for HSA, but my results have not yielded a value close to that of the accepted value. We have seen various values for HSA Free Thiol content on the web, but most are around 0.7 mol/mol. I am getting 0.4 mol/mol, which is relatively far off (considering that there is one free thiol in the molecule). I am using the Ellman's Assay with a cuvette - as we also had issues using a plate based method (which I hope to end up at, as using the cuvette method using way too much sample). I am mixing 25 uL Ellmans Reagent (4 mg/mL in 0.1 M sodium phosphate, 1 mM EDTA pH 8.0) with 125 uL HSA (at 30 mg/mL) and 1.25 mL of the sodium phosphate. I am blanking the instrument with the ellmans reagent, water, and the sodium phosphate - could this be an issue? Should I blank with just water and then run with all of the components?

I'm unsure if it is my calculations or if there is an issue with the assay (I have been attempting this for months - and have bought new reagent to ensure that is not the issue).

For the calculations my steps are simple and as follows:

1) Determine the final concentration of protein in the cuvette (30 mg/mL X (125 uL volume of protein/1400 uL total volume) = 2.68).

2) Take this value (2.68) and divide by the MW of HSA to get 40.301 X 10-6.

3) To get the concentration of free thiol, divide the absorbance (.201) by the extinction coefficient (14150) to get 14.205 X 10-6.

4) Divided the free thiol concentration (14.205) by the final concentration of protein (40.301) to get a mol/mol value around 0.35. I know this is incorrect.

Any help would be greatly appreciated. I have combed over almost every website and every post on Research Gate to find a solution - I have been trying this for three months, almost daily, and still have yet to find a solution. Any guidance would be great, thank you all!