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pure protein (?) but two bands on SDS-PAGE

pure protein (?) but two bands on SDS-PAGE - Protein Forum

pure protein (?) but two bands on SDS-PAGE - Protein Forum


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Old 07-25-2006, 01:14 PM
Rhiannon Evans
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Default pure protein (?) but two bands on SDS-PAGE



Hi, I have cloned the gene for my protein into two pET vectors, one with an
N-term His-Tag, one without. When I purify my recombinant protein from E. coli
BL21(DE3)RIL Codon Plus cells via affinity chromatography (methotrexate or
Nickle column) I get two bands close together at approximately the correct
molculer weight for the target protein on SDS-PAGE. I have tried to denature the
protein in 8M Urea, and reduced any disulphide bonds with mercaptoethanol and
TCEP, but still get two bands. MALDI-TOF Mass Spec shows a peak at the correct
molecular weight only. I am thinking there has been some sort of post
translational modification?? Any suggestions would be appreciated. Cheers :-)

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