GST fusions

[R. Woodward]

Dear all
  
 
  
 




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 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
-doc??
 Robert W
 Email rw200 at cus.cam.ac.uk                                            .
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