problems with GST-peptide binding after Sarkosyl lysis
P.J. van Santbrink
santbrin at CHEM.LEIDENUNIV.NL
Tue Mar 18 06:01:11 EST 1997
Dear fellow netters,
I'am trying to isolate my GST-fusion protein from my lysate with
beads but it seems that the fusion protein won't bind.
I've lysed the transformed bugs with Sarkosyl (2%) according the
Frangioni and Neel article published in Analytical Biochemistry vol.
210, 179-187 (1993) and though I
got a significant amount in the supernatant fraction, I'am not able to
isolate the fusion protein from the supernatant fraction (or from the
pellet fraction): when I lysed
the bugs with the addition of Triton X-100 I was able to isolate the
fusion protein but
in very small amounts.
- lyse with lysozyme and 2% Sarkosyl
- centrifuge, add triton X-100 (3-5%) to solution, vortex
- add glutathione beads and incubate at 40C (overnight, shaking)
- centrifuge, save sup, wash beads 5 times with PBS + 0,1% Triton
X-100 + 150 mM NaCl
- elute with 20 mM glutathione + 2% N-octyl glucoside in 50 mM TRIS
pH 8,5 (pH is correct after addition of glutathione): incubate 2
hours, 40C, shaking
- bring monsters on 10% SDS PAGE gel and perform silver staining
After the silver staining, all my fusion protein was in the first saved
fraction (= non bound protein).
Question: what is the reason my fusion protein doesn't bind to the
beads? Should I add more Triton X-100 to the sarkosyl lysate before I
add the beads?
I hope someone can give me any advise because though I'am able to get a
large part of
my fusion peptide in the supernatant fraction (which is quite an
achievement, I think),
I also would like to get the protein purified (to perform binding
You can E-mail me privately or via this medium
Peter van santbrink
Division of Biopharmaceutics
Leiden (the Netherlands)
E-mail: santbrin at lacdr.leidenuniv.nl
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