I have gram negative bacterial samples (different mutants of the same organism) on which I am willing to perform a global proteomic analysis to find out deferentially expressed proteins. But we are having problems to characterize membrane proteins which are crucial for organism to produce hydrogen (my mutants are different in terms of hydrogen production). Can anyone suggest me a method that will increase the number of membrane proteins in my samples. Thanks in advance.
Mecit.
Hello Mecit.
We have found increased membrane proteins using Jo Loo's "eFASP" technique as outlined in this paper:
http://pubs.acs.org/doi/pdf/10.1021/pr4010019
Good luck!
Peter
Dear Mecit,
How is your sample preparation? Membrane proteins are known to be hard to solubilize. Are you including detergents in the sample extraction/preparation step?
Probably, the most important factor in enriching the number of membrane proteins identified will be to do a parallel experiment of membrane and cytoplasmic proteins isolated by eg., differential centrifugation.
Good luck,
Hediye
You can try phase seperation based on the protocol by Bordier C. 1981. http://www.ncbi.nlm.nih.gov/pubmed/6257680. Membrane protein seperation worked well in our hands but we have still to publish the results.
Thank you so much for your suggestions. I am really grateful.
We are using a differential centrifugation method to isolate both cytoplasmic and membrane protein isolates, however, the LC chromatographs of the samples are really bad that we decided not to move on to MS/MS. That's way I am trying to find an alternative method.
The method that we used can be found in the following link. http://www.sciencedirect.com/science/article/pii/S0167701206001242
For soluble proteins; we lysed the cells with bead beating, add urea and thiourea up to 7M and 5M concentrations respectively. Add DTT, desalt the sample and trypsinize them overnight. Desalt them again with column. Determine concentration and run LC.
For insoluble proteins; we lysed the cells with bead beating, and centrifuged at 100krpm for 10 minutes. Take the pellet and sonicate it, centrifuge at 100krpm for 5 min. The pellet was resuspended in a solubilizing solution that contained 7 M urea, 2 M thiourea, 1% CHAPS in 50 mM NH4HCO3, pH 7.8, and an aliquot of 194 mM DTT solution.
To simplify the process, Pierce produce a membrane protein kit which saves a lot of sample prep time and works on the basis of phase seperation. You might also want to use surfactant removal columns to clean up the cell membrane protein part of the product.