Help: EGF-receptor purification
hubert at cigale.u-strasbg.fr
Fri Jun 10 04:56:22 EST 1994
We are trying to prepare solubilized lectin-purified EGF receptors from
A-431 cells for binding and kinase studies. From the litterature we
picked up a method involving solubilization in Triton X-100,
chromatography on Wheat Germ Agglutinin-agarose, and elution in
acetylglucosamine + MgCl2 + glycerol. On the day of purification, EGF
binding and EGF stimulation of receptor autophosphorylation work OK. Our
big problem is that after a few days in the -80! freezer, EGF binding and
kinase stimulation decrease and eventually are completely lost. We can
still detect autophosphorylation, but no stimulation by EGF, and
autophosphorylated receptors appear as oligomers upon non-denaturating
electrophoresis analysis, with very little monomer.
Are there any tricks to allow for conservation of active EGF-receptors
for a few weeks ?
Is there a better partial purification procedure ?
Or is it better use freshly prepared receptors for each experiment ?
Thanks a lot for any tips.
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