Why HEPES and folic acid in my lysis buffer? - (Dec/31/2007 )

Dear All,

Happy New Year!

I have one question here:

I am purifying DHFR (dihydrofolate reductase). I know that DHFR reduces folic acid to its active form (tetrahydrofolate). But i don't know why i should use folic acid in the lysis buffer to purify this Enzyme DHFR. Also i don't know why i should use HEPES instead of Tris. I use Ni-NTA beads as the first step of purification followed by Q column. Since Tris is better than HEPES for Q column, can I use Tris in the lysis buffer instead of HEPES?

Any reply would be greatly appreciated!

I believe FA binds to active center and protects the enzyme during the purification process.

Tris is fine for Q-column.

although i don't know for sure if it is a significant problem for this use, tris is a chelator (like edta, maybe not as strong) and may interfere with protein binding to nickel.

Dear mdfenko and genehunter-1,

Happy New Year and thank you so much for your thoughtful reply!

What buffer do you use for Ni-NTA resin? For me, I use Ni resin twice during the DHFR purification. My boss asks me to use HEPES and Folic acid (FA) for the first Ni column and Tris for the second Ni column, which i really don't understand.

As genehunter-1 said, FA protects the enzyme during the purification process. i don't know why no FA added during the rest of purification. No FA added for Q column, gel filtration and the second Ni column.

Thanks a lot for your reply in advance!

The folic acid may still be there, if it remains tightly bound.