Protein Extraction - (Jun/12/2006 )

I have got my expression in E.Coli but the probelm is its soluble form. I extrct my protein with two diffest methods.
First Just 200mM Tris and 300mM Nacl and band is very low.
Second I extracted my protein through 200 mM Tris and 300 mM Nacl and 0.1 percent SDS and I got a sharp band as compared to ths simple Tris and Nacl Buffer.
Please suggest me what can i do now. I have to determine the structure of protein and vibrational spectroscopy. I take expression at lower temperature as well but the result is same.
So waht you suggest If I uses 0.1 percent SDS and i got a sharp band then any problem with protein or not.
withbest regards
from
samita

If your protein is insoluble, slow down the expression rate. That means temperature (which you have done...how low have you gone?), but also IPTG concentration. Try different levels and see if any of them give you better solubility.
If not, have you tried solubilising the inclusion body with ~1M urea? Often, low levels of denaturant are all that's needed to resolubilise the whole thing without totally denaturing the protein.
Have you tried gentle lysis, say freeze/thaw? Have you tried sarkosyl (0.1 to 1%) in the lysis buffer?
Keep trying, something is sure to work .

I tried .1 mM, .5 mM , 1mM and 16 and 30 tempertaure but no too much difference I have seen.
My protein is expressing with out IPTG and I put glucose . Tell me its better to use glucose or not use glucose at all. I use freeze thaw method with Tris and Nacl and Tris Nacl 0.1 percent SDS and
band is very sharp in NaCl, Tris and 0.1 % SDS. I have to do structural study, tell me some good information about the purification of proteins and I will upload the picture and you can have a better idea for suggestion.
regards

What about the RPM of shaker. If low down temperature then also low down RPM. I mostly use 200 RPM and Induce for 5 hour at higher cell mean 0.7 OD at 600nm. RPM with lower temperature matters or not or keep at 200 RPM.

you can use bugbustre or sonication to obtain you protein.
IF the protein expressed in inclusion body, you can lower induction temperature to 15 degree, and induction overnight, RPM is not the critical parameter (200 rpm normally);
In my experiments, the concentration of IPTG optimization is not signaficant.
Good Luck!

if nothing else helps try refolding after denaturating extraction -
never done it before but try this link for further infos:

REFOLD

Good luck

Hi, I've been struggling with an insoluble protein also. However, I started using Artic Express cells from Stratagene and they worked very well. The only problem is that you have to induce for 24 hours at 11 C.

hope that this helps.