2 replies to this topic

[zienpiggie]

Hi all,

I am just wondering if anyone has ever extracted protein with simple phosphate buffer pH 7.4 with 1 mM EDTA, and I freeze thaw 3 x to lyse them and run them for western blotting? Is that acceptable at all? I have read abcam's guidelines for lysing cells and it appears that RIPA or NP40 buffers are being used with 30 minutes agitation in chilled condition. I don't know how different it would be if I start lysing my cells with RIPA buffer or NP40 buffer instead..

Thank you

[bob1]

It will work for the particularly soluble proteins.  The detergent steps help solubilise (some of) the poorly soluble proteins and degrade fats etc. that are found in the membranes.

Freeze/thaw cycles can also result in the degradation of the proteins in the samples.

[zienpiggie]

Thanks Bob1. I am trying RIPA buffer now to lyse my cells.