Hepes concentration

Hello,

I know that 10mM HEPES y a right concentration. I would like to know if 5mM Hepes it also right.

Thank you,

Papillon

Hi Papillon,

You can use 5 mM Hepes.
The Hepes in the buffer is used as a buffer compound to manage the pH.
If you lower the concentration it is possible that the pH will deviate from what you want.

You can also use other buffer compounds like Tris or phosphate ...

Why do you want to lower the Hepes concentration?

Arnoud

Hi Arnoud,

I want to use 5mM Hepes because the research had study the interaction by another techniques and she would like to reproduce the same buffer conditions. I think she use Hepes alone. It isn't 150 mM in NaCl. But for her it is also important to have a good pH reproducibility. The interaction should be study a pH 7.4 and pH 8.5. The analyte change its conformation between this 2 pH. I think above pH 8.
If a 5mM Hepes concentration it isn´t advisable, please tell me and I´ll use Hepes 10mM?
Can you gave me your Hepes recipe to check mine?

Thanks,

Papillon

Hi Papillon,

I use in general 10 mM Hepes pH 7.4, 150 mM NaCl + 0.005% P20.
The Hepes as a buffer component, the NaCl to make the buffer more fysiological and to diminisch some non-specific initeraction and the P20 (tween-20) also to reduce non-specific binding. Sometimes I add calcium or EDTA depending on the application
That said, there are a lot more other buffer systems around wich perform equally well. You must use the buffer which is suitable for your application.

I gues you want to have a stable protein at pH 7.4, so use the 10 mM Hepes and adjust the pH to 7.4.

To observe conformational change per se, I would not recommend SPR. Use Circular Diochrism instead.

Arnoud