problems with GST-peptide binding after Sarkosyl lysis
Fri Mar 21 14:51:17 EST 1997
On 18 Mar 1997, P.J. van Santbrink wrote:
> 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
I had exactly the same problem myself a few years ago - as far as I can
recall we used to leave the lysate for 30 mins after the addition of
triton - possibly increasing the "removal" of the sarkosyl.
Maybe more Triton as well.
You may want to try less sarkosyl - do you really need 2% - I used 0.75%
for a large protein I worked on for a while - I also used it for binding
assays - seemed to work ok.
Hope this helps a little bit - but to be honest - His tagged proteins are
far easier to purify (my opinion) - less fuss.
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