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Western blot using FACS cells

Western blot using FACS cells - Protein Forum

Western blot using FACS cells - Protein Forum


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  #1  
Old 01-03-2008, 05:19 PM
Melissa Mazan
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Default Western blot using FACS cells



Hi all -
I am trying to identify proteins using WB on cells that have already
been through FACs. We make a single cell suspension from lung, bring it
on ice to our core facility (about 45 minutes away) and then bring the
sorted cells back to the lab in media on ice. About 4-5 hours in all.
The sorted cells I receive range from 1200 cells/ 250 ul to 1x10 to the
7th cells. I wash the cells at slow speed in PBS (4C) when I receive
them, then extract protein using M-Per from Pierce. I'm not having much
luck with samples that have less than a million cells. Any suggestions?
Many thanks - Melissa

--
Melissa R. Mazan, DVM, Diplomate ACVIM
Associate Professor and Director of Sports Medicine
Tufts Cummings School of Veterinary Medicine
200 Westboro Road
North Grafton, MA 01536
508-887-4589

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Old 01-16-2008, 07:14 PM
Dr Engelbert Buxbaum
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Default Western blot using FACS cells

Am 03.01.2008, 13:19 Uhr, schrieb Melissa Mazan <[Only registered users see links. ]>:



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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