Hi ... I've scraped cancer cell line without throughout the media ... all the suspension transfered to a tube and stored at -80 C.
How can i extract protein from this one please any ideas or protocol
Thanks
The link is the protocol for the Western blot, Hope it is helpful
http://www.abcam.com/protocols/general-western-blot-protocol
Freeze thaw of cells leads cells to rupture and release their contents in to the media collected with which leads to improper protein collection. Better perform another expt.
If you want to analyse the entire protein profile you can extract protein by lysing with RIPA buffer as rightly mentioned. If you want to analyse proteins in different compartments of cell then you can use buffers with varied salt concentration and fractionate cytosol, nuclear and chromatin bound proteins. There are various protocols available which you can go through.
Hope it helps!
Thanks all for your answers
I will do new experiment and in time of cell collection i will use the RIPA buffer for protein extraction and go ahead my project ... i will not store the cells at -80 C ...
Hi Saad,
Don't forget to add sufficient proteinase inhibitor, most of the protein is fragile if there is present of proteinase
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