I am working on a heterodimeric protein expressed in same E.Coli, two different lysis buffers are used for their purification. i.e.
50 mM NaH2PO4, pH 8, 300mM NaCl, 10mM imidazole, and
50mM Tris-Cl, pH 7.3, 10mM NaCl, 0.4mM DTT, 1mM EDTA, 1.2mM PMSF, 10% glycerol.
which one should be preferred if i would use only one buffer?
First of all, phosphate shoulb be used around pH 7 and tris around 8. You can use either one, the selection depends on what you want and how are you going to proceed with purification. The first buffer implies that you will use imac for purification and that you will not perturb the dimer. The second buffer will not work for imac. DTT will break disulfide bonds, EDTA and PMSF are added to inhibit proteases (these things can also be added to the first buffer).
but these buffers with same pH values are used in sample papers and they go directly to Anion exchange chromatography mean no IMAC. and my protein is hexameric and dodecameric in equilibrium, so what you suggest.
Everybody doing things wrong does not make it right. Anyway, the first buffer will not work for iex due to the high concentration of NaCl, moreover phosphate should not be used for anion exchange chromatography. What is the size of the monomer?
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