I wanna compare the cell membrane proteins in two different cell types of the same bacteria. They are not overexpressed by any expression systems. The two cell types just belong to two different generations.
First I wanna isolate the membrane proteins. Then, do proteomic profiling (quantitative and qualitative).
Any suggestions on the protocols for gram-positive membrane protein isolation and proteomic analysis would be highly appreciated.
Gram positive cells do not have an outer membrane.
Pellet the cells from the culture with a centrifuge. Treat the cells with lysozyme to degrade the peptidoglycan, then sonicate or French Press the cells to break up the membranes.
Collect the membranes by ultracentrifugation. Resuspend the membrane pellet in a buffer. Pellet again to wash off residual cytoplasmic proteins. Use the pellet for your proteomic study.
More details can probably be found in journal articles relevant to your subject.
Adam B Shapiro Is treating the cells with lysozyme is sufficient? As it is Gram-positive, should I use lysostaphin also?
One more clarification. I want to get the whole cell protein (both soluble and insoluble). After ultracentrifugation, can I take the supernatant as soluble and the pellet as insoluble fraction?
Lysostaphin is recommended for lysing Staphylococcus aureus cells specifically. Lysozyme can be used for other Gram positive bacteria.
After ultracentrifugation, can I take the supernatant as soluble and the pellet as insoluble fraction?
Yes.
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