GST purification multiple band problem - GST purification problem, multiple band (Nov/15/2006 )

Hi,
I'm trying to purify a soluble GST-tagged protein (82kDa) and am having the problem of eluting multiple bands after column purification. I have tried expressing in various bacterial strains ie. BL21, BL21(DE3), Rosetta Blue as well as lowering the temperature for expression to 18 degrees overnight. I have also tried all types of solbule purification methods to help with degradation and non-specific protein sysnthsis and still no luck. Any suggestions would be greatly appreciated. So far my current protocol goes as follows:
- Transform purified plasmid DNA into BL21 (DE3), grow overnight starter culture from colonies
- Diltute stater 1:100 and obtain OD at 0.6-0.8
- Express overnight at 18 degrees using 0.1mM IPTG
- Solubilise bacterial pellet using sonication in TBS with 1% Triton X100, protease inhibitors
- Filter sample and load onto a GSTrap 1ml column
- Elute using 10mM reduced glutathione in TBS

EDTA, PMSF, DTT, lysozyme during solublisation and even batch purifcation using GSH beads have all been trialed and still no difference.
Any help please..

Regards

Brett

hi

are u using pGEX vectors?

i read in the manual of pGEX vectors column elution part that sonication should be kept to minimum time but maximum result u wil have to optimize it,, time should be kept near 10 seconds of course that will depend on how much volume u will use for sonication and the type of sonicator

if the sonication is more than that is enough it will cause other proteins to elute out with the recombinant or cleaved protein .

did happen to me once, had to optimize it ,, maybe ur seniors in the lab knows the best condition for the particular sonicator for a particular volume? that will make it easier for u

oen more probabilty is that ur protein has different forms or dimers or polymers if the size of the other bands are higher than ur protein and if its lower , it means some sort of degradation propably due to over sonication or other technical aspects .


hope it helps

regards
laxmi

Thanks for your reply,

Yeah the vector is pGEX-4T3. I have also tried without sonication and still the problem of multiple bands occurs. Even when I do sonicate it is performed on ice with short bursts for an on-off time period of 2 minutes. My protein of interest shows up at 82kDa with a strong band at 50kDa and 3 smaller ones following that. I'm thinking my next step is to perhaps use size exclusion but I was hoping to get rid of the other proteins without resorting to an additional purifcation step.

Cheers

Brett

am not sure if this also applies to GST purifications, but for histag purifications you can add some imidazole to the washes. Do u think low concentrations of glutathione in your washings will clean ur band?