[Arabidopsis] protein precipitation during dialysis

Y Jiang via arab-gen%40net.bio.net (by jiangyqcn from hotmail.com)
Wed Nov 5 17:37:50 EST 2008


hello, colleagues,
 
I met a problem with a plant transcription factor (TF) protein expressed in E.coli. The TF protein was expressed and purified through anti-His resin from inclusion body (as it was not expresssed in the supernatant, but in the inclusion body). It was later dialyzed against a buffer (25 mM HEPES/KOH at pH 7.5, 40 mM KCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT and 30 mg/L PMSF) at 4°C. This dialysis buffer is copied from a ref describing a different Arabidopsis TF using almost the same expression vector and purification system. However, the protein precipitated during dialysis and after centrifugation, I quantified the protein concentration of supernatant and found that it is very low(almost negative calculating from the standard curve, Bradford assay). So most proteins went into the the pellet after spining. I need to get some biochemically active protein for an assay. Does anyone know how to solve this? The pI of the TF is 10.3.  Is it worth diluting the protein before dialysis? Or should I use a different dialysis buffer with a lower pH? 
Your feedback is greatly appreciated.
 
Thanks.
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