Dear All,
My Lysis buffer recipe is 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100 and 5% glycerol.
I would like to use this buffer to lyse whole cell, except nuclear membrane, which I will lyse with RIPA buffer later.
Most nuclear fractation protocols are use of 0.05-0.1% TritonX1-00/NP40. I am not sure if 1%TritonX-100 can be too high and might lyse nuclear membrane. Moreover is 150mM NaCl high enough to lyse nuclear membrane?
Thank you
Cheerio,
1% Triton is very high and yes 150 mM NaCl is quite high too. With that buffer you may most likely get whole cell lysis.
You need a hypotonic buffer to lyse the membrane leaving the nuclear membrane intact. I am copying an example below. Hope this helps.
Hypotonic Lysis Buffer (HLB)
10 mM Tris, PH 7.5
10 mM NaCl
0.3 % NP40
10% Glycerol
Not sure about the salt (that could dissociate the proteins and thus help to disintegrate the nuclear membrane), but the 1% Triton is high for sure.
From my experience with extraction and nuclear isolation. I would recommed first to use hypotonic buffer to get the intact nuclei first and then use second buffer to lyse the nuclei completely.
Nuclear isolation:
First resuspend the cells in 1 mL ice-cold hypotonic buffer (10 mM HEPES pH 7.4, 5 mM MgCl2, 10 mM NaCl) with 1X protease inhibitor for 30 min at 4 °C, and then homogenize 50–100 strokes in a Dounce homogenizer using a tight fitting pestle to rupture the plasma membrane. The soluble cytoplasmic fraction can be separated fromthe insoluble nuclear fraction by centrifugation through a 200 μl 40% sucrose cushion centrifuge at 800g for 20 mins in a swinging bucket rotor. You will get nuclei as pellet. (if you need highly pure nuclei you need use two different concentration of sucrose cushion and you have to centrifuge at 32000 rpm (105 000 g) for 1h). If you need that protocol, i can email you)
Extraction of nuclei:
Since the membrane protein does not solubilise that easily use 7M urea and 1% triton to solubilise them completely.
In the below paper you can see,
http://www.sciencedirect.com/science/article/pii/S000527361400217X
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