Dear all, I'm currently working with Avibacterium (close family with Haemophillus). I want to compare the expression of an outer membrane protein across different growth phases. There's no MAb available for this protein and producing it in-house is not an option.
A previous work shows that this protein is 600 kDa and whole-protein comparison (via SDS PAGE) with a knock-out mutant for the gene shows no other proteins are of this weight. We don't have an ultracentrifuge to do membrane protein purification/fractionation. So I want to do whole-cell protein extraction and compare the amount of this protein via SDS PAGE and gel-image analysis.
Currently available protocol is to collect the cells, pellet, resuspend in PBS, add SDS PAGE sample buffer, boil, then load onto the gel and run the electrophoresis. Is there any suggestion on how to maximize the protein yield during the extraction, especially because my objective is not merely to detect but to quantify (relatively between various time points)? Do cells from different growth phases have different susceptibility to the lysis buffers (thus affecting the yield)? Thank you in advance.
This would be preety lengthy explanation, I suggest you to go through some book chapters in pubmed or springer protocols. Yes you have to harvest at different times and test where you will get maximum yield. Outer membrane proteins are difficult to extract, you have to test various ways to extract your protein of interest like optimizing detergent of your choice. It would be better to start with non ionic detergents in your case. I think, you might find an ultracentrifuge in nearby lab and do the sucrose gradient.
I also suggest finding an ultracentrifuge to separate the total membranes from the cytoplasmic proteins. This will greatly enrich the sample (pellet) for membrane proteins. It may not be necessary to purify the outer membranes by sucrose gradient, however. You may be able to dissolve the inner membrane with N-laurylsarcosine, then pellet the outer membranes. Resuspend the outer membrane pellets in a small volume of buffer and measure the protein concentration. This will allow you to compare the amount of the outer membrane protein of interest across samples normalized for total outer membrane protein.
Article Two-step purification of outer membrane proteins
Finally dissolve them in SDS-PAGE sample buffer. If the protein is a 600 kDa monomer, you will need special gels.
Article Efficient Electroblotting of Very Large Proteins Using a Ver...
Cells at different stage of growth have different susceptibility to sonication with or without a detergent. Prefer addi g protease inhibitor mix in the cell lysis buffer, and you need an ultrafiltration step for separation of membrane proteins.
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