I am trying to lyse C6 cell lines which are cancerous glial cell lines of rat origin. Kindly let me the best way to achieve cytosolic and nuclear protein?
I take it you're trying to collect nuclear and cytosolic protein fractions for western blot or some other downstream application? I use a nuclear lysis kit from Millipore (product number 2900) that does the job quite easily. There are probably many other commercial alternatives, or homemade buffer recipes that will work.
Basically, after collecting the cells you lyse them but leave the nuclei intact. Spin down the nuclei and collect the supernatant (your cytosolic fraction). Resuspend the nuclei in another buffer (nuclear lysis buffer), lyse them, spin down the membranes, and collect the supernatant as your nuclear fraction.
For the actual lysis, the kit recommends passing the suspensions through a syringe, but we've also successfully used a few pulses with a probe sonicator.
Good luck!
thank you Maa'm...can you please let me the standardized amplitude and on off time of the sonicator probe for mammalian glial or neuronal cell lines?? we actuly make our own lysis buffer using various components. we have never tried kit yet.
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