I am facing a problem during extraction of total proteins from cell lysate. I am using cell lysis (RIPA) buffer with standard composition as described in scientific literature. But every time my samples goes slimy which creates trouble for me, for my next experimentation.
If someone have solution regarding to this problem, please share with me.
Hi Ravi
how much cells you harvest??? you need at least 300ul of RIPA for confluent 10cm plate , you need to adjust your lysis buffer volume to your cells number/weight.....
i think you add less RIPA to your cells as you need....
also you can to do sonication for your cell lysate.......
another note is to make sure that your RIPA composition is right
I would sonicate your sample with a probe, 12µA for 30 second pulses on ice water should do the trick. The DNA in your extracts will make it viscous but the sonication will shear your DNA.
Hello Mr. Sleman Bsharat
I am using 1ml RIPA buffer for 100mm culture dish having ~70-80% cells. And composition and pH of components are as per protocol describes by various researchers. But I didn't perform sonication. Hope this will work.
Thanks for your valuable suggestion.
Hello Mr. Edward Grahme
I didn't perform sonication. I will perform this. Thanks for your valuable suggesion.
I would either sonicate the sample or add benzonase to your lysis buffer when lysing the cells. This has worked very sucessfully in the past for us. Doing this will degrade or shear RNA and DNA respectively and help reduce the viscosity of the sample.
Thanks Mr Gary James Spencer..... I will try this also. Thanks to all for giving their valuable time and suggestion to my query.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction