I've been looking at protocols to do cell fractionation. I want to be able to do Western Blots on both the cytosolic and nuclear fractions. Currently I'm not seeing any of my protein in the nuclear fraction which is strange. Does anyone do this on a regular basis? Can you point me to a protocol which works reliably for you?
Hi Adam, here is a simple and nicely working procedure (final extract w/o DNA):
• Wash cells with ice-cold PBS
• Scrape the cells in a small volume of ice-cold PBS
• Spin 13.000 rpm, 1 min
• Add 1.3 ml RSB to the cell pellet. Incubate 5 min on ice
• Spin 13.000 rpm, 1 min
• Remove sup. Add 0.5 ml RSB (with DTT, Na3VO4, & Protease Inhibitors)
• Homogenize with a dounce glass-glass homogenizer (B-pestle). 30 Strokes on ice.
• Spin 13.000 rpm, 1 min
• Supernatant = Cytosol, Pellet = Nucleus
• Resuspend the pellet in 3 vol Buffer C and incubate on ice for 20-30 min
• Spin 13.000 rpm, 2 min
• Supernatant = nuclear extract
• Store samples frozen (-80)
RSB
10 mM Tris, pH 7.4
10 mM NaCl
6 mM MgCl2
Buffer C
20% Glycerol
20 mM Hepes, pH 7.9
420 mM NaCl
1.5 mM MgCl2
0.2 mM EDTA
Good Luck!
Hi Adam
Here is the method that I use, which seems to work well. (http://www.jbc.org/content/285/12/8743.long)
Nuclear and cytoplasmic fractionation was performed by a modification of the method described previously (14). Briefly, cells were trypsinised (2-3 wells of a 6 well plate) and washed twice in ice cold PBS and lysed on ice for 15min in 100 microlitres of cytoplasmic lysis buffer (10mM HEPES pH 7.4, 10nM KCl, 0.01mM EDTA, 0.1mM EGTA, 2mM DTT, 5mM Na¬2VO4, 20mM sodium β-glycerophosphate, 0.1% NP40 and ‘Halt’ Protease inhibitor cocktail (Perbio, Chester, UK). Nuclei were sedimented by centrifugation and the supernatant containing the cytoplasmic fraction removed, to which Urea and SDS were added to a final concentration of 2M and 2% respectively, and the samples denatured by boiling for 5min.
The nuclei were then washed in 1ml of cytoplasmic lysis buffer to remove any contaminating cytoplasm, and re-sedimented. The nuclei were then lysed in 100μl of denaturing lysis buffer (2% SDS, 2M urea, 8% sucrose, 20mM sodium β-glycerophosphate, 1mM NaF, and 5mM Na¬2VO4). Genomic DNA was sheared by passage through a Qiashredder column 6,000 rpm 2 minutes in a benchtop microfuge (Qiagen, Crawley, UK), and denatured by boiling for 5min. Protein concentrations were determined by the BCA assay (Pierce/Perbio, Chester, UK).
To determine even loading and purity of the extracts I did westerns on them using IGF1 receptor (cytoplasm) and Lamin B (nucleus).
Good luck
Hi Adam, here is a simple and nicely working procedure (final extract w/o DNA):
• Wash cells with ice-cold PBS
• Scrape the cells in a small volume of ice-cold PBS
• Spin 13.000 rpm, 1 min
• Add 1.3 ml RSB to the cell pellet. Incubate 5 min on ice
• Spin 13.000 rpm, 1 min
• Remove sup. Add 0.5 ml RSB (with DTT, Na3VO4, & Protease Inhibitors)
• Homogenize with a dounce glass-glass homogenizer (B-pestle). 30 Strokes on ice.
• Spin 13.000 rpm, 1 min
• Supernatant = Cytosol, Pellet = Nucleus
• Resuspend the pellet in 3 vol Buffer C and incubate on ice for 20-30 min
• Spin 13.000 rpm, 2 min
• Supernatant = nuclear extract
• Store samples frozen (-80)
RSB
10 mM Tris, pH 7.4
10 mM NaCl
6 mM MgCl2
Buffer C
20% Glycerol
20 mM Hepes, pH 7.9
420 mM NaCl
1.5 mM MgCl2
0.2 mM EDTA
Good Luck!
You can fractionate the cell components based on theattached protocol:
HI Ivan,
I tried your fractionation protocol, but I seem to have cytosolic contamination in my nuclear fraction. Could there be a wash step missing? Should I try washing with Buffer C before continuing on to the nuclear extraction step?
Thanks for your help!
Use NE-PER kit (Pierce) or try the attached protocol using home made buffers. Good luck.
Hi Biliana,
There will always be some cytosolic contamination with this protocol. To minimize it one has to remove as much of the cytosolic supernatant as possible. Washing once more with Buffer C may disrupt your nucleus, resulting in lower yield. If you need very clean nuclear fractions you should use a more stringent protocol (e.g. sucrose gradient fractionations).
Hope this helps!
I agree with Ivan. According to a paper, it is common to get 10% cytosolic contamination after nuclear extraction, even after thorough washing.
If you want a really good subcellular fractioning, use the Calbiochem kit: (Ref. 539790, 418 euros)... It worked for me, I saw clean cytosolic, membrane/organells and nuclear fractions after using it...(no tubulin and CoxIX in the nuclear fraction!!) The kit is expensive but it is worthy, in my oppinion
Good luck!
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