I'm extracting protein from human fibroblasts in RIPA buffer. I will try 10,000 xg to start off, but I know some cell types require lighter centrifugation than others.
Hi Sian,
I typically centrifuge protein lysates for 5 minutes at full-speed (14 000 rpm, table top centrifuge). I am curious to see what others think, but I highly doubt that you would be able to precipitate any proteins with this short centrifugation step, unless it wasn't soluble to start with or is a protein with unusual properties. If you want to be 100% sure that there is no debris (for instance, for ELISA), you can repeat the centrifugation step with the supernatant.
Good luck!
Hi,
the general protocol in our lab states the following:
after collection of your lysate, incubate for 30 min on ice.
Spin down for 10 min at 13.000rpm
transfer supernatant to new eppendorf tube.
Good luck!
Hi,
I feel that both the above protocol is fine, and should work well. Just one suggestion, see that there is sufficient RIPA buffer for complete lysis.
Regards
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