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
glutathione
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.
Protocol:

   -  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
studies).

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



More information about the Methods mailing list