We have a protein of 40kDa size with his tag. purification with Ni-NTA column showed impurities. when gel filtration was run after ni nta column chromatography, again same bands appeared on SDS PAGE though gel filtration showed only one peak.
Hi there,
SEC is not resolutive enough to separate proteins with close MW. IEC (resolution based on overall apparent charge) or HIC (based on hydrophobic properties) are more recommended if it is the case.
hey daniel,
Imidazole gradient was used as following:
wash1: 20mM Imidazole, wash2: 60mM Imidazole, wash3: 100mM Imidazole, elution: 150mM Imidazole.
after wash fractions, all the elution fractions have the same kind of impurities.
Impurities bands are lower than protein in size. Gel filtration is producing similar pattern in SDS gel.
Hi again,
An other possibility is that your protein undergoes some cleavage and that the cleaved protein remains under its native state (peptides issued from cleavage interacting tightly so the cleaved protein behaves as the non cleaved protein during SEC). However peptides are efficiently separated during SDS/PAGE (denaturing condition)
Hi Jai,
I would recommend you to elute over a gradient. If not pure after that, you can add an ion exchange purification step. As Dominique said SEC is not resoluive enough to separate proteins with close MW. Even if the impurities are lower on a SDS PAGE gel, they can be as dimer, trimer.... in your protein mixture and thus elute in the same peak with your protein over a SEC.
-Imidazole gradient over 10-20 column Volum
-ion exchange chromatography
Good luck
Hi Jai
I agree with Dominique and Dacleu, In this case SEC is not resolutive, after affinity chromatography you must use ion exchange or hidrophobicity chromatography. It´s suggested load the sample with low imidazole concentration to avoid unspecific binding. Another tip is in all teh buffers to maintain 300-500 mM Nacl (just for Ni-NTA). Good luck !
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