[Protein-analysis] Re: Western blot using FACS cells
Dr Engelbert Buxbaum
(by engelbert_buxbaum from hotmail.com)
Wed Jan 16 14:14:01 EST 2008
Am 03.01.2008, 13:19 Uhr, schrieb Melissa Mazan <melissa.mazan from tufts.edu>:
> I am trying to identify proteins using WB on cells that have already
> been through FACs.I'm not having much luck with samples that have less
> than a million >cells. Any suggestions?
A mammalian cell has about 10 um diameter (1.2e-9 cm3 volume or about 1e-9
g weight given that the density of cytosol is slightly above 1). Protein
content about 10% by weight = 1e-10 g of protein. Thus your 1e6 cells
would contain approx 100 ug of total protein, which is a reasonable to
smallish load for a minigel. Distribute that over, what, 30 bands and you
have just enough protein to detect an average band with Coomassie. Those
with less than average amounts of protein perhaps with silver.
Smaller gels (e.g. PhastSystem) may give you a slightly higher
sensitivity, but miracles you should not expect.
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