I treated some cancer cells with inhibitors, lysed them with laemmli buffer (containing protease and phosphatase inhibitors) by scraping on ice. They were left on the rotor for 16hrs in a cold room to allow proper lysis. After centrifuging at 16000xg I notice that there are large sediments at the bottom of the tubes including the control (no treatment). I am worried because when I carried this same protocol previously I did not notice any thick (mucoid) white sediment has anyone else notice this?
Are you referring to Laemmli sample buffer? If so its usually SDS precipitating out in this buffer. Part of the reason people use LDS is because it doesn't precipitate out at low temperatures like SDS. Maybe it got colder than usual? Or there was less protein to soak up SDS? Gently reheating the samples may put the SDS back into solution, they might get a bit more denatured though. Be aware that too much heat can reduce S-S bonds even without added reducing agent.
Hi Lily, this is not unusual. I have seen this many times.When you treat cells with SDS, the cells lyse and release their DNA and the entire prep looks like snot.
Get some syringes with low bore needles to suck the lysates through and dispel to shear the DNA. ~10 times repeat should get rid of this problem.
Thanks to you all. There was definitely less protein to soak up SDS.
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