Can anyone suggest a protocol for extraction of nuclear and cytoplasmic proteins from cell line without kits and detergent solution (NP-40)...
Can I use Nuclear Extract (NE) buffer (HEPES [20 mM] pH 7.9, NaCl [0.4 M], EDTA [1mM], Glycerol 25%, Protease Inhibitors 1x (add just before use)?
you can see: Andrews & Faller, 1991, NAR 19(9) p2499 for a protocol
You will be able to use this along with mechanical shearing, such as dounce homogenisation. You will not get membrane-bound or cytoskeletal proteins though.
you can see: Andrews & Faller, 1991, NAR 19(9) p2499 for a protocol
Juan Carlos Polanco,
sir can i use Triton x-100 nstead of IGEPAL CA 630, ..
Supernatant with CLB buffer will give you soluble cytoplasm without detergents. Supernatant with TSE buffer (with IGEPAL CA 630) will give you cytoplasmic proteins associated with membranes and cytoskeleton. TSE Pellet will be the nuclei. I have not tried TRITON-X100 with the TSE buffer but it might work similarly to IGEPAL CA 630. However, I don't know if can affect nuclei integrity. You can give it a try and test it.
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