Protein sticks to Ni beads - Protein doesn't elute from the column/batch etc (Apr/09/2009 )

Hello,
I've expressed my 6-His-tagged 59kDa protein using baculovirus expression in Sf21 cells. It's expressing, soluble and binding to Ni-IDA beads very efficiently. However, I cannot elute it even with 1M imidazole. NP-40 doesn't work either. When I check on SDS-PAGE after 1M Imid, it's still sticked to the Ni beads. I tried to stripe the Ni from the beads, however it's not promising either . Does anyone know how to deal with these tenacious proteins.

Thank you

This is what I do:

1. Try Talon beads

2. Strip the Ni off by using EDTA in elution bufffers.

But it may not be the best way to go about it.

Best,
TC

GeneTurk on Apr 10 2009, 03:21 AM said:

Hello,
I've expressed my 6-His-tagged 59kDa protein using baculovirus expression in Sf21 cells. It's expressing, soluble and binding to Ni-IDA beads very efficiently. However, I cannot elute it even with 1M imidazole. NP-40 doesn't work either. When I check on SDS-PAGE after 1M Imid, it's still sticked to the Ni beads. I tried to stripe the Ni from the beads, however it's not promising either . Does anyone know how to deal with these tenacious proteins.

Thank you

Hi

It seems Your protein is highly/strongly bound to the resin. this type of problem can be overcome by using the resin otherthan Ni, i mean use cobalt or cupper or zinc resins which are highly works well like Ni-NTA or Ni-IDA the differnce is protien binding is little week so you can easily elute the protein with Imidazole.

If you want to use only Ni-IDA then obvoiusly you have to use EDTA in the elution buffer and once you have completed your elution , dilute/dialye the elution sample with required buffer( reducing the EDTA and Ni concentration)

Hope it may work

All the best
Regards
Sudhakar

GeneTurk on Apr 9 2009, 12:51 PM said:

Hello,
I've expressed my 6-His-tagged 59kDa protein using baculovirus expression in Sf21 cells. It's expressing, soluble and binding to Ni-IDA beads very efficiently. However, I cannot elute it even with 1M imidazole. NP-40 doesn't work either. When I check on SDS-PAGE after 1M Imid, it's still sticked to the Ni beads. I tried to stripe the Ni from the beads, however it's not promising either . Does anyone know how to deal with these tenacious proteins.

Thank you