vary the pH slightly...

I have tried pH range from 7.5 t0 8.5 but all in vain. I used cysteine standards of different concentrations and these are showing such a gradation with Ellman's reagent but no color with BSA.

Could you use a cystamine as a mediator

try prepare a new solution, with different concentration and vary de pH

@Karl Please convey how to use cystamine and what is its mechanism.

cystemine used to react the protein to amide bond formation .do you have this type of reactions cont ion for concentraction and pH variation as repeat the make up solution to try to do.

I think that, sometimes free SH of cys 34 blocked during purification step, so you should first liberate it to get result with ellman's reagent

Concerning your investigation, I may argue that the conditions you are using in oxidizing BSA are strong enough to convert all surface sulfhydryl groups in sulfoxides.

I would try 1) Ellman's test on untreated BSA (blank reaction); 2) on BSA denatured before and after the oxidation treatment. Anyway you may give a look to: Anal. Bioanal. Chem. (2002) 373 : 266-276.

dissolve Ellman's reagent by dissolving in appropriate buffer of 0.1 M sodium phosphate, pH 8.0, containing 1 mM EDTA.if colour doesnot produced perform experiment by increasing the pH.

1. Ellman reagent staining BSA when performing dithiol-thiol exchange reaction. In another words, the -SH functional groups on BSA needed to react with the -S-S- dithiol group on Ellman reagent to have one half of the Ellman reagent attach to BSA via forming BSA-S-S-R and RSH to generate a yellow color. If your BSA formed intramolecular dithiol with it own -SH under certain pH condition or in our previous treatment, then you may not be able to get a inter-molecular dithiol-thiol exchange reaction, neither the color.

2. If your BSA have been contaminated with mercaptoethanol or DTT then you color but don't expect concentration dependent response.

@Kuang So what is the most desirable pH to get the color.

I don't know. I did this exp years way back to my earlier days as an analytical chemist. However, it is an easier experiment to set up finding the optimal pH. Still, you may possibly encounter in the following circumstance; 1. degradation of DTNB(but you did it with the addition of cysteine the color appear so DTNB is okay.) 2. your BSA been experienced strong oxidation condition so the -SH functional groups are not accessible for thiol-disulfide interchange.(get a little amount of BSA from your neighborhood lab and get the test redone) 3. the concentration of your BSA is too low to get an appreciable color change.(inc amt of BSA to see if the number of moles of -SH is comparable to the concentration of your cysteine test?)

Thiol-disulfide interchange reaction is a broad pH spectrum chemical reaction, I really don't think pH really matter that much aroung slight alkanline pH? Believe me.

@Kuang Thanks a lot for such an explanation. I'm getting color with Cysteine and the only thing I didn't do is changing BSA supplier.

Regards

Hi Bilal,

Please check your cysteine concentration in number of moles and back convert this into BSA number of moles(assuming BSA mol wt is 68,000) I believe then you will get the color you expect. Nevertheless, you may find that you are using quite a lot BSA then you originally imagined. Not enougn BSA in the testing solution is what I think you're coming onto.

Dear Bilial,

Did you solve your problem? May I ask how?

Thanks

Dear Anabela

Yes I did. I used to dissolve Ellman's reagent in distilled water, you have to dissolve in pH8 buffer

Well, that is not my problem...I already dissolve Ellmans in PB pH 8...Did you write any article with the protocol? Can you send me? How did you prepare BSA?

@Anabela I haven't communicated any paper right now.

you need to use 1mg/mL BSA

I dissolved 2mg/ml of BSA in water. No color developed with the Ellmans reagent neither before or after reduction with NaBH4...

Since Ellman's reagent reacts with free SH groups, you can react cysteine with Ellman's to check whether there is any problem with the reagent itself

I already did it. I tested with cysteine and with cystine, reducing it to cysteine with NaBH4. It worked fine. I will test if the problem is not using a denaturant, or if I need to pre heat the BSA.

Dear Bilial

I have the same problem with HSA and think maybe it's the question of protein concentration because I mixed 50microliter of protein solution (1mg/ml) into 1ml of DTNB solution(0.5mM), prepared in 100mM phosphate buffer. I wonder if you could find a solution or not?

Meysam