Why hepes buffer turned yellow - (Apr/11/2008 )

The buffer contains: 200mM Hepes, 1mM DTT, 1mM EDTA. Hepes free acid was used when making the buffer and pH was adjusted to 6.8 with NaOH. One day after the buffer was made it turned slighly yellow and two days after it is prominently yellow in color. I wonder why this is happening and if the buffer is still OK for further use.

I guess that is the problem of DTT. As a strong reductant, DTT is unstable even at 4 oC. Maybe you need to add fresh DTT from 100 mM DTT stock solutions that was kept at -20 oC or lower.

Thank you for the suggestion.I smelled the yellow hepes buffer and it indeed does not smell very much like fresh DTT. But I made a 100mM phosphate buffer using the same DTT stock the same day and it seems nothing went wrong. I think I have seen reported buffers using DTT with 50mM Hepes so possibly Hepes does not react with Hepes at this concentration. I need around 200mM Hepes with DTT and the buffer needs to be stable for several weeks at room temperature. I wonder if this is possible?

I saw many protocols describing the buffers that contains DTT prepared by such a way: store X fold DTT stock solution in aliquots at -20oC... Just prior to use, add DTT to a final concentration of x mM. Although I am not sure that your hepes buffer turned yellow is caused by DTT, I suggest to add DTT to the desired concentration from a stock solution just prior to use the buffer (including the phosphate buffer). Please see the informations about DTT's half-life at varied conditions from Sigma, http://www.sigmaaldrich.com/sigma/product%...et/d9779pis.pdf
http://www.sigmaaldrich.com/sigma/product%...et/d9779pis.pdf