I have tried to purify membrane protein in HEPES buffer but still there are some other buffers there. Which buffer and other biochemical parameters should be followed for better protein induction and purification. For membrane protein purification, which buffer is the better choice so that I can maintain my membrane protein folding and membrane hydrophobic part?
I agree with Melissa, you have to choose your detergent more than your buffer. During induction you can improve your expresion using a E. coli strain with high ratio of membrane , like C41 or C43 instead of, e.g., BL21. Regarding TritonX100 and NP40,they are not the same: NP40 has an octyl tail and triton a metilbutiril tail and sometimes they are not equivalent in order to solubilize proteins.
What end purpose are you going to us the protein for? Functional assays? Crystallization? Something else. For functional assays and crystallization I would test the ablitity of different detergents to solubilize your protein away from the rest of the cellular material. I would try one or two detergents from several chemical families like:
OG and NG, DM and DDM, LDAO, CHAPS and CHAPSO and compare the solubilization to SDS-PAGE sample laoding buffer containing SDS. Consider the SDS solubilization to be the effective 100% solubilization detergent. Take samples of your cells and lyse the cells and solubilize for 2 hours at 4 degrees C with very gentle agitation (slow enough to not make any bubbles). Take out a gel sample and label it "before spin" and then take a sample and spin it hard (20,000 x g for 30 minutes) withdraw the supernatant and label that the "after spin" sample. Run a gel on the samples and compare the before spin to after spin, the detergents where the before and after spin are equal are the best solubilizing detergents
Hello Timothy, your link and suggestions are fantastic. It really have nurtured my ideas regarding crystallography.
Well, I think you could try RIPA Buffer. This buffer solution works very well extracting this kind of proteins.
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques