SURVEY response summary: GST-fusion elutio

T. S. Grewal sasgrewl at reading.ac.uk
Mon Feb 13 09:40:01 EST 1995


**************************************
John
rjl6n at uva.pcmail.virginia.edu

writes:
Anyway, I've generally had very good success eluting GST fusion proteins
from beads. These fusions are typically in the 85 -120 kDa range (getting
them soluble is another story). However, I had trouble eluting one of my
fusions until I realized that I had changed the elution conditions; on
this occasion, I was eluting in a modified PBS-type buffer at about pH
7.3. However, when I switched back to my normal elution buffer of 50 mM
Tris (pH 9.0!) + 20 mM glutathione that same fusion protein eluted
readily. So, I would suggest that another explanation might be that your
pH is marginally too low and is OK sometimes, but doesn't work other
times. Oh, and I've used both Pharmacia and Sigma beads with equal
success.
***************************************

I found that if you increase the conc of GSH for elution
to above 10 mM, you need to increase the conc. of
the Tris buffer to 100mM to insure that the pH stays at 8.0 or 9.0.

Also the addition of 150 mM NaCl helped incease recovery of my GST
fusion protein (47 KDa).
You could also try adding 5 mM DTT to the elution buffer, this 
may help elution of larger fusion proteins.

GOOD LUCK.

T.S.GREWAL





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