What are people’s preferred methods for preparing lysates for protein analysis (Western, IPs)?
What method do you use to homogenize the sample? Beads? Sonicator?
I’ve only done a few westerns, but we use a sonicator
We are currently using RIPA lysis buffer (https://www.emdmillipore.com/US/en/product/RIPA-Lysis-Buffer-10X,MM_NF-20-188) for all our tissues and glass pestle (see image below) for DRGs, and a tissue grinder for SC and brain.
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We have the beads and we would like to try them, but we never had time for this testing and the pestle works well. Hopefully, next year we can have some summer students to try the beads.
I used beads back at Duke. They work! And they’re consistent. You just need the apparatus to shake the beads, which can be a bit pricey. But especially for consistency and throughput, beads are great.
For denaturing gels for westerns, we use protein lysis buffer (with protease and phosphatase inhibitors) added to tissue and sonicate.
For IPs from tissues, I believe it would be the same.
For collecting cells cultured from plates, I used to add western lysis buffer containing tris and SDS directly to wells, then scrape and pipette into tubes. This is a rough method though, it breaks down bilipid membranes so the lysate is very goopy. A couple of freeze-thaws from -80 and quick spins usually clears the lysate of DNA.
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