I am doing nuclear and cytosolic fractionation. what im using is 0.1% NP40 in buffer A having composition of 50 mM b-glycerophosphate, pH 7.3y1.5 mM EGTAy1 mM EDTAy1 mM DTTy0.1 mM Na3VO4 with all protease inhibitors.
I recommend TNES buffer.
for preparation 100 ml TNES buffer mix:
5 ml Tris (1m) pH 7.5 at 25'c
8 ml Nacl (5m)
20 ml EDTA (o.5 M)
10 ml SDS (0.5%)
Top up to 100 with dH20
vortex and filter.
Do you need to have nuclear and cytosolic proteins in the same fraction or you want to separate them on the basis of solubility?
Generally mild lysis buffer (I used,50 mM HEPES,
125 mM NaCl, 0.1 mM phenylmethylsulphonyl fluoride, 0.2% NP-40, pH 7.5) to isolate soluble fraction; you have to carefully regulate the centrifugation as well (300g,10 min at 4deg). The remaining pellet can be lysed by RIPA to obtain the insoluble(nuclear,membrane,organelle) fraction.
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