My protein has His-tag, Mw 27kDa and pI=10. Which purification step is suitable to purify secretary protein form the Pichia BS culture media, pH 5? Do I need to adjust pH to 7-8 then following with IMAC or I need to concentrated the protein by Centrifugal Filter 10 kDa MWCO then adjust pH and IMAC? Thanks, in-advance😉
Hi there,
For IMAC, imidazole ring of histidine has to be deprotonated. So you have to work at pH above imidazole pKa (which is about 6.5).
You could also concentrate your protein first on catex column and then elute by increasing both pH and salt concentration, both of which are desirable for IMAC.
I normally adjust pH of the culture media to around 8 with solid tris and add NaCl to 0,5 M and whatever Imidazol concentration I need. And off course filter before running IMAC. In most cases I wash with a buffer with lower salt concentration (150 mM) before elution so that I can continue with IEX with only 3-5 X dilution.
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