sonication protocol - I know we have a pinned topic, I just need a protocol (Apr/01/2008 )

hello all,

I know we have a pinned topic up there about cell lysis, but I was wondering if someone could give me any protocol for lysing mammalian cells.

I want to extract total protein from HeLa and MCF-7 cancer cells to run a Western blot for Bcl-2 protein.

I made a RIPA buffer but it didn't give me good bands on SDS, so I want to try sonication but I have no protocol.

thanks
Aidin

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sonication is good if you do not like to use detergent lysis;

in your case, better separate higher amounts of protein or enrich Bcl-2 by immunoprecipitation...

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yes, that is why I want a protocol. I thought I only need PBS, but apparently I need a solution before I put the probe inside the tube.

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Dear Aidin,

Buffer: Tris HCl 50mM
Antipain 10ug/ml
Pepstatin 0.5uM Note: These protease inhibitors reduce proteolytic attack.
DTT 0.1mM
Leupeptin 0.5uM
PMSF 0.1mM
pH 7.5

Sonication: Probe sonicator NOT sonic waterbath. We use a Soniprep, made by MSE.

J774 murine cell line: 3 bursts of 5 second ON ICE, with 25 seconds intervals in between.........15 Amplitude microns power. Sonication causes the cell suspension to heat up, doing this on ice REDUCES the possibility of protein denaturation in this step.

IT IS VERY IMPORTANT TO OPTIMISE THE SONICATION FOR EACH CELL LINE i.e. check under the microscope for fractionation of the cells.

This method has given me beautifully clear bands on westerns looking for iNOS enzyme expression. It also gives me an active cytosolic enzyme that we use for kinetic/co-factor studies.

Hope this is useful.

Kindest regards.

Rhombus

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Thank you very much, I was about to just use PBS. wow.

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