protein purification - his-tag protein purification (Oct/07/2004 )
i am working with a 20-27 kda protein. actually they r different deletion mutants.
all of them have a his-tag at N-terminus. and they exist as inclusion bodies.
once i lyse the cells using lysozyme, the suspensin becomes so slimy and it continues even after adding Dnase and Mgcl2. Even after washing it with detergents like NP-40 and triton X-100, it remains the same.
Finally when i dissolve the inclusion bodies in 8 M urea some of the mutants go into the solution but few not....they still remain slimy.
purification using Ni-Probond column is 70% ok, and get high molecular weight proteins.
cananybody suggest me some thing?
I usualy double my volume of Lysis buffer to make sure it's not to slimy. I spin my lysate at 25,000rpm at 4 degrees for 30 minutes and incubate just the supernatant from my lysate with the beads. I've never use the urea since I usualy work with native form of the protein I don't know if you should put it in before of after the spining the prep.
Your proteins should be in solution unless they are with the TM domain.
Hope it help a litle bit
try to use Benzonase (Novagen) at 1ul/ml resuspension buffer instead of DNase. I got rid of all the slimy stuff.