Hi, At such concentrations, it might be necessary to concentrate the medium. Otherwise you are likely to be working well below the KD of the affinity beads. At 0.5mg/L, 50KDalton gives you 10nM. The KD of Ni-NTA should be around 1uM, according to published SPR data http://www.sciencedirect.com/science/article/pii/S0003269797923265. This means the majority of your protein will stay in solution even at equilibrium. One of my colleagues does use Ni-NTA beads for their secreted mammalian protein, which produces at >10mg/L. And he still concentrates it at least 10x.
Zhijie From: Jerry McCully Sent: Monday, April 23, 2012 9:37 PM To: [email protected] Subject: [ccp4bb] minimum protein concentration for NI-NTA column Dear All; This is a sort of naive question about the NI-NTA affinity purification. Is the Ni-NTA column from GE health able to capture proteins at 0.2ug/ml to 0.5ug/ml? IF not, then it is necessary to concentrate the mammalian expression supernatant. Thanks a lot and best regards, Jerry McCully
