Why Ni-NTA beads became clumped after incubation? - Intensive Clumped (May/31/2010 )
What bothered me is the Ni beads became clumped after incubating with E. coli lysis of His tagged protein.
I don't know if it is because the sonicated E. coli lysis is too cencontrated (100ml medium of E. coli resuspened and sonicated in 7ml buffer) or because of the binding of his-tagged protein or because there is any step else that I haven't realized.
Why donīt you explain your experiment with more detail please: ml of resin against these 7 ml of feed, buffer (pH), the absortion is in batch or your resin is paqued?, are you using a FPLC?. I guess you are not making any capture step before de AF, arenīt you?, which kind of resin are you using?, FF or HP, no metal chelators?, no PEI?
paramyosin on May 31 2010, 11:44 PM said:
hi,I concentrated 100ml of E. coli and resuspend in 7ml buffer,sonicated,now the lysis is very sticky.then I concentrated the lysis in 12000rpm for 30min and pipet the sticky supernatant into EP tube with Ni NTA beads which is washed once with lysis buffer already.then after 2hour of incubation ,the beads became clumped.
so what is capture step?and AF?
what is FF HP or PEI then?
well,metal chelators,for Ni NTA,it is.