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western blot--protein concetration - too dilute?? (May/16/2008 )

I think my cell lysate is too dilute to do western blots. I have only about 2ug/ul in 300ul. Is there a way to concentrate the proteins? Please help!

-maria123-

QUOTE (maria123 @ May 16 2008, 12:56 PM)
I think my cell lysate is too dilute to do western blots. I have only about 2ug/ul in 300ul. Is there a way to concentrate the proteins? Please help!
Check out these protocols, not all of 'em sleep.gif
http://search.vadlo.com/b/q?sn=158621799&a...k=tca&rel=0
..

-cellcounter-

QUOTE (maria123 @ May 16 2008, 01:56 PM)
I think my cell lysate is too dilute to do western blots. I have only about 2ug/ul in 300ul. Is there a way to concentrate the proteins? Please help!


simpliest is precipitation (Wessel & Fl├╝gge-protocol or TCA), and then boiling in Laemmli buffer

-The Bearer-

do you mean 2ug/mL or 2ug/uL (ie 2mg/mL)? Cause 2 ug/uL is a pretty good concentration for western blot. I typically run 20-30ug for westerns, so 300uL would be plenty of protein.

The easiest way as already mentioned would be to precipitate your lysate (TCA is one of the most common ways). In the future you may want to grow more cells and/or lyse in a smaller volume, that should increase your concentrations.

-smitity-