This is not normal. If anything, the color increases rather than decreases over time. I doubt the issue is caused by Tris, though this is not the buffer of choice for DTNB assays, nor is the pH of 7.5. Typically, you would use 0.1M phosphate, pH 8.0. Your Tris buffer is a bit weak at 25mM too, which could be borderline if the cysteine standards are acidic (often they are). Do you have anything else in the buffer or standards? If not, I'm pretty sure a switch to the phosphate buffer mentioned above will fix the problem.

Nick Gee I'm trying this assay with Tris buffer because it is the most common formulation buffer we use. The buffer also contains 200mM NaCl. Could the salt interfere?

No, it won't be caused by salt (as long as it is only salt) and there is nothing obviously wrong with Tris or pH 7.5 buffer for that matter; it's just that a higher pH and phosphate buffer is more commonly used.

Are there any other components in any other solutions that you are using? Is the tris buffer composed of only Tris and NaCl, for example? If so, then there has to be something odd about the formulation of the standards or with the combination with the assay buffer, and I'm drawn to the idea of acid as the culprit, esp. with your relatively low buffering capacity. What did you make up your cysteine standards in? Trace metals ions in any buffers (potentially arising from Tris or salt) would also be of slight concern.

If you need to continue with Tris buffer, I'd suggest that you use 100mM rather than 25mM (pH 7.5 is okay, but 8.0 would be better from a buffering perspective). Also add 1mM EDTA to the buffer. (If you add EDTA to a solution already made up, re-check the pH after doing so).

The point of these suggestions is to ensure that the TNB anion is at the right pH for a strong color and to prevent reoxidation of the TNB anion back to colorless DTNB.

Good luck and let me know how it goes.

I agree with Nick Gee

1. This is unusual. I've never heard of it before.

2. It's probably due to re-oxidation, which may be facilitated by acid pH and traces of transition metal cations.

3. Switching from Tris to phosphate-EDTA will mop up those pesky dications.

4. NaCl shouldn't cause any problem, as long as it's reasonably pure.

5. Cys is often obtained as a HCl salt, which produces a robust acid pH. While 25 mM buffer does not seem terribly low, as your Cys standards probably do not exceed 2 mM, buffer is cheap, and there is no good scientific reason to use such a low concentration of buffer in your assay.

I would only add:

6. At least double-check your buffer recipe for proper concentration, make a fresh batch, and re-calibrate your pH meter before adjusting pH.