z-suldan at SKI.MSKCC.ORG
Tue Nov 22 09:15:12 EST 1994
>what glutathione concentration is your colleague using? around 10 mM
>should work. but 10 mM gsh would significantly reduce the pH of a
>phosphate buffer. so try using something like tris. check the pH AFTER
>addition of glutathione and should be around 7 to 8.
Just to emphasize this.... I did a little titration about a year ago
with my gst fusion protein and found that as the pH drops, the elution from
the glutathione beads drops significantly. So much so that even an elution
pH of only 7.0 gave me 50% elution compared to a pH of 8.5.
2 more hints.
Don't use the "free acid" form of glutathione to make your elution buffer.
10mM will give you a pH down in the 3-4 range and is a pain to pH. We now
use Sigma G-4251 and it is much eachier to pH.
You might also try glutathione -Sepharose beads (as opposed to agarose
beads). Someone else in our lab found that her ELUTED yield was
TREMENDOUSLY higher with the sepharose than with the agarose.
Tri-Institutional MD/PhD Program - Department of Cell Biology and Genetics
Memorial Sloan Kettering Cancer Center / Cornell University Medical College
Replies to: Z-Suldan at ski.mskcc.org or ZSuldan at Stud.Med.Cornell.edu
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