whole cell lysis buffer

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whole cell lysis buffer

Postby geniaco » Jan 16 2017 6:59 am

Hello All,

I would like to lysate my cells to get a protein localised in the cytoplasm.

Could you suggest me agood one or even a recipes ?

Thank you.
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Re: whole cell lysis buffer

Postby relaxin » Jan 16 2017 12:56 pm

50 mM Tris HCl, pH 8.0 and 1% Triton X100.
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Re: whole cell lysis buffer

Postby geniaco » Jan 16 2017 3:40 pm

Thank you for your quick reply.
Does this bufer require a loading dye after lysing cells?
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Re: whole cell lysis buffer

Postby relaxin » Jan 17 2017 1:02 pm

If you want to analyze the lysate on SDS gel, the best lysis buffer is nonreducing sample buffer without dye (NRSN-Dye) which is 60 mM Tris HCl, pH 6.8, 10% glycerol, 2% SDS. The omission of reducing agent and dye in the buffer allows you to determine the protein concentration. You can lyse your cells (without culture medium) in a flask or dish with 500 ul of NRSB-Dye containing 1% protease inhibitor cocktail. Then transfer the lysate to a microfuge tube. With the tubes submerged in ice-water, sonicate for 2 cycles at 15 sec/cycle. Then centrifuge at 10,000 rpm for 15 min at 4 C to remove cell debris.

After protein determination by BCA method, you can add 50 ug protein in a microfuge tube, adjust volume to 31 ul with sample buffer, then add 2 ul of 0.125% bromophenol blue and 2 ul of 1 M DTT, heat at 95 C for min. The sample will be ready for SDS gel electrophoresis.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
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Re: whole cell lysis buffer

Postby geniaco » Jan 17 2017 1:04 pm

Thank you so much! :D
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