rw200 at cus.cam.ac.uk
Thu Jan 26 09:34:12 EST 1995
I have recently been expressing ion channels or truncated portions of ion channels as GST fusion proteins. Thses express tovarying but exceptable levels, as judged by SDS-PAGE of cell lysates. Ido have problems when it comes to binding my fusionproteins to the glutathione sepharose column when only approx 30% binds, I should not be saturating my beads and I have tried various methods of solubilisation from triton x100, sarkosyl, urea (after renaturation), cholate, SDS before mafing my lysate 1-2% triton
before I add to equilibrated matrix, I have also altered the incubation time (15mins-16h) and temperature 4-22oC.
I aslo have a problem in eluting the fusion protein which does bind, routineelyonly achieving approx 10% recovery, I have tried altering; ionic strength, glutathione concentration (10-20mM), adding detergents, 1.2M urea, increasing the temperature and duration of the incubation etc... The GST controls show similar characteristics though not nearly so bad. I realise that these are common problemsand maybe characteristic of individual proteins but can anyone out there offer some advice to a frustrated post
Email rw200 at cus.cam.ac.uk .
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