Hi Parastou,

We perform this assay in our lab for GMP testing of free thiols, so yes this will give you a correct result assuming:

-TCEP has been removed or doesn't interfere with the assay (I have no experience with it)

-you are using accurate extinction coefficients to determine your protein concentration and conjugated free thiol concentration

-there are no impurities absorbing at either wavelength

We use a high concentration of guanidinium chloride to ensure access to the free thiols, I'm not sure if that step is necessary to your protein. We expect 3 free thiols, and get results in the range of ~2.9 to 3.05.

I would recommend measuring your samples 15 min after Ellman's reagent is added and minimize light exposure to the Ellman's reagent both prior to and subsequent to addition. I did repeat measurements of the samples and by the 4th measurement at 60 min saw almost 10% reduction in absorbance. I haven't done a better experiment yet to see if time causes degradation or light exposure.

If you want any protocol details, let me know. Otherwise, best of luck to you!

Thank you so much Dear Jeremy Pronchik for your reply. I appreciate more details regarding calculating the number of Cys based on the sample absorption number.

Sorry that this will read a bit weird, I'm copying and pasting sections of our protocol:

Upon reaction with reduced cysteine sidechains (free thiol groups), a yellow species is produced that can be quantified by absorbance at 412 nm (A412). The reaction can be accelerated by the addition of a concentrated chaotrope such as 8 M Guanidine HCl or Urea, but the molar extinction coefficient will be adjusted from ε412 = 14,150 M-1cm-1, DTNB’s ɛ, to 13,700 M-1cm-1, TNB’s ɛ, because the extinction coefficient of the thiolate is slightly dependent on ionic strength. This is relevant when thiols are determined in guanidinium chloride where the extinction coefficient at 412 nm drops to 13,700 M-1cm-1. It is important to note that the ɛ of TNB is different in different solvents at A412.

We prepare:

1. guanidine hydrochloride, 8 M

2. Ellman's reagent buffer: 100 mM Sodium Phosphate, 1 mM EDTA, pH 8.0 (I made a mistake and adjusted to pH 7 once, there was almost no conversion at 15 min)

3. Ellman's reagent: 12 mg in 10 mL Ellman's reagent buffer

4. We dilute our proteins to ~7 mg/mL, that should be more or less good for your protein

Measurement 1: A280 and A412

50 μL sample (~7 mg/mL) or buffer

200 μL Ellman’s Reagent Buffer

750 μL 8 M Guanidine HCl

Measurement 2: A412

add 50 μL Ellman's reagent

Calculations:

protein concentration:

A280,corr = A280,sample - A280,buffer

[protein] = A280,corr/ε280

free thiol concentration:

A412,corr = (A412,sample,pre-Ellman's - A412,buffer,pre-Ellman's) - (A412,sample,post-Ellman's - A412,buffer,post-Ellman's)

[free thiols] = (1.05*A412,corr)/13700

free thiol ratio = [free thiols]/[protein]

Just make sure your extinction coefficients have the same units of course. That factor of 1.05 in the [free thiols] calculation is to adjust for the fact that protein concentration is measured with a solution volume of 1 mL, and thiol concentration is measured with a solution volume of 1.05 mL.

If that isn't clear, let me know and I'll try again.

Thank you dear Jeremy Pronchik I got your point. Actually, I drew a standard curve by L-Cys and could determine my sample Cys concentration based on the standard. The concentration is by micromolar; i want to know how to convert this molarity to Cys number! how can I report how many Cys are available in my sample!?

do you know how to measure your protein concentration by A280? If you don't know your protein's extinction coefficient, then you can calculate it using this website:

https://www.biomol.net/en/tools/proteinextinction.htm

If you don't know the sequence of your protein, you can try to find it on the PDB:

https://www.rcsb.org/

see attached pics for each if they're new resources for you.

Then ratio the concentration of free thiols in micromolar to the concentration of your protein in micromolar. It's what the calculations above are doing, but I see you're using a different procedure. You can use those calculations, but change the extinction coefficient for the thiols to ε412 = 14,150 M-1cm-1 if you didn't use guanidine hydrochloride.

Dear Jeremy Pronchik Thank you so much for detailed explanation.