I have cloned, expressed, and purified an hypothetical protein (13 KDa) from S. aureus using pET 25b expression vector. Protein contains 1 cysteine residue which may accounts for dimerization. To prevent it I started my purification using 1mM DTT in lysis and chromatography buffers but still it turns out as a dimer as per analysis via superdex (gel filtration G-75) and SDS- Page experiments. Is there any way to prevent its dimerization and purify it as a monomer product?
Hi Shehzaib
First have you Run your protein in native phage and conform weather the protein is monomer or dimer or tetrameter. If u conform that your protein is Monomer than you can try the buffer contains the BME(β-mercaptoethanol) up to 12mM Concentration instated of using DTT it will work. All the best.
This protein seem to be a dimeric in native conditions. If you want monomer for some reason then try aklylating cys residues to prevent dimer formation.
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