I am performing a phosphor-proteomics project and need 10-20 mg/ml protein in 100-200 ul buffer. I am using an 8M Urea buffer and sonication (15-20 2sec bursts) for lysis. I performed a test run using 1.25e6, 2.5e6 and 3.75e6 cells, each in 25 ul buffer, but only was able to obtain up to 2.5mg/ml. I am thinking that this is largely due to the low volume, which is suboptimal for sonication. I would like to believe that lysing more cells in 100-200 ul buffer would suffice, but I was hoping someone has experience with protein extraction from lymphoid cells.
Hello!
So you are basically aiming at 1-4 mg of protein for your project, which sounds like a reasonable starting point for a phospho-proteomics experiment.
From my experience of doing proteomics with primary, activated/blasted CD8 T cells, I know that I get 60-80 ug of total protein out of 1x10^6 cells if I lyse them in 8M urea (Which is much more than you'd get for example from naive T cells, where you can expect 30 ug per 10^6 cells). You seem to be getting ~20 ug per million cells which doesn't strike me as too far off (I don't know much about the size of pre-B cells; as a reference activated CD8 T blasts are approx 8-9 um in diameter and naive T cells approx 6 um). The small volume might indeed not be optimal for lysing/sonicating cells but can be good enough depending on the sonicator.
Either way, you will have to scale up considerably to get your minimum of 1 mg of protein: From the calculations above you'd need approx 50x10^6 cells to get 1 mg of protein. Lymphocytes are much smaller than tumour cell lines like HeLa, so in terms of cells numbers you'll need much, much more starting material.
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