I am trying to purify an insoluble GST-tagged animal membrane protein (70kDa) and I am having problem of eluting multiple (3) bands after column purification. These are having band size of slightly less than my protein (~5kd). I have tried expressing in various bacterial strains i.e. BL21, BL21(DE3), Rosetta Gami, P-LysS as well as lowering the temperature for expression to 16 degrees for 6h. I have also tried all types of soluble purification methods to help with degradation and non-specific protein synthesis and still no luck. I solubilise bacterial pellet using sonication in PBS with 0.3% Sarkosyl, 1% Triton X100, 1.mM CHAPS and protease inhibitors.
May be stringent washings may help you.Increase the volume of your wash buffer.
Hi! Asif..can you tell me by which column you are purifying your protein??? is it Ni-NTA matrix or GSH??
First of all, have you cheked by western-blot if your contaminant band do correspond to your protein ( degraded forms of your protein) or if they correspond to other contaminat proteins.
If they are different proteins, you can perfom additional purification steps to remove your contaminat protiens and if they correspond to your protein degradation, you should work on improving your production or lysis step.
HAve you tried to use French press to lysate your cells instead of sonication. Strong sonication procesdures sometimes can damage your protein.
This is not an uncommon problem for inclusion bodies. Bacteria do have a system for clearing these unwanted artefacts of overexpression that include proteases. If you have a good antibody maybe you can perform a induction time course to see if there is optimal point when you can harvest. Otherwise you could consider an additional c-terminal tag, or as suggested earlier an additional purification step.
Dear Colleagues I need some video to describe using of GST rap HP-GE column step by step. could you please help me ? thanks
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