I want to enrich the membrane proteins from lyophilized bacteria, I used the sodium carbonate method, sucrose gradient method. But I couldn't identify 5-10 proteins, even less sometimes by LC-MS. Is it possible to identify more membrane proteins from lyophilized samples? my approach is right? i shall be thankful to your suggestions.
Instead of starting with lyophilized whole bacteria, you could start with fresh bacteria. Break them open with sonication or other method, centrifuge at low speed to pellet unbroken cells and large cell fragments, then centrifuge the supernatant at high speed to collect the membrane fraction. Resuspend the pellet in buffer and pellet again to wash. This is an enriched membrane fraction that can be used for further analysis. Run SDS-PAGE, cut out bands, digest with protease, and identify bands by LC-MS and comparison to proteome.
Hi Ravi, I don't have experience purifying membranes from lyophilized bacteria. It might be OK. It's always a good idea to work with fresh material if it's available.
Out of curiosity: are 100% sure the problem is in the enrichment step?
Going from crude material to LC-MS analysis, there are tens of steps which could "go wrong".
If you can share your complete procedure, we may help you troubleshoot it.
I read the protocol.
it is basically the carbonate fractionation method.If I have to guess, the problem is the centrifugation step after ice cold Na2CO3. It may very well be that you are not "precipitating" your proteins.
Alternatives to this method go basically in one direction:
Detergents: here you can basically read "kits". You can use combinations of soft and hard detergents to enrich your proteins. Pros: detergent are the best at solubilizing these proteins. Cons: they are the worst enemy of mass spectrometry.
You might want to check for cleavable detergents.
Alternatively, you may want to try protocols such as this one
http://www.ncbi.nlm.nih.gov/pubmed/23334993 (never tested, but I am quite curious about it).
One last thing. There are different methods for inner and outer bacterial membrane proteins.
Read this discussion for further review
https://www.researchgate.net/post/What_is_the_best_protocol_to_obtain_the_membrane_protein_fraction_from_bacteria
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