Attempting affinity purification.
Vector backbone is pGEX-6P1. Insert encoding my protein of interest is 2.4 kb in length. Composition of lysis/binding buffer is 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA, 1 X protease inhibitor cocktail and eluting with 10mM reduced glutathione. My system is working as I can elute GST proteins from vector transformed cells. But GST tagged protein of interest is not binding to beads (Glutathione agarose beads) as I cannot detect any protein bound to beads by SDS-PAGE. I have tried various binding conditions, eg. Binding Buffer with 10 mM DTT or 1% Sarkosyl or 0.03 % SDS, but no improvement.
You need to check your expression levels and solubility of your protein. This will involve optimizing the strain, growth and induction conditions. It is possible the GST tag is not good for your protein, in which case you could put it at the other end of your protein or change to a MBP or His tag. It might also not be eluting from your beads so you should boil the beads in SDS and run that on your gel as well, to see if you have protein.
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