GST fusion protein won't elute

Cornelius Krasel krasel at alf.biochem.mpg.de
Tue Nov 15 03:58:20 EST 1994


T. S. Pillay (tpillay at ucsd.edu) wrote:
> He has tried to purify the GRB2 SH2 domain but he finds that the protein
> does not elute off the beads with glutathione(GSH).  I know this sounds
> weird but has anyone experienced a similar problem with this or any other
> fusion protein.  He gets good induction of expression when looking at
> whole cell lysates but very poor yields with purification.  When he 
> takes the GSH-sepharose beads after elution with GSH and boils it in
> laemlli buffer- he finds "tons" of the fusion protein indicating that the
> fusion protein is sticking avidly to the beads and not eluting off.

Sounds like unspecific binding to the sepharose to me. A simple way
to test if the GST part is functional is the enzymatic assay for GST. It
measures the conjugation between 2,4-Dinitrochlorbenzene (DNCB) and GSH. The
product absorbs quite strongly at 340 nm.

Pipet together
1 ml	200 mM Potassium phosphate, pH 6.5
100 ul	20 mM GSH
790 ul	aq. bidest.
10 ul	sample
100 ul	20 mM DNCB in EtOH

and measure extinction at 340 nm. There is some spontaneous reaction between
DNCB and GSH, so you have to make the control without your protein.

But even if the GST part is functional it could still be possible that the
SH2 domain part leads to unspecific binding to the column.

--Cornelius.

--
/* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany  */
/* email: krasel at alf.biochem.mpg.de                 fax: +49 89 8578 3795  */
/* "Science is the game you play with God to find out what His rules are." */



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