sucrose gradient for protein fractionation???

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minylim
minylim's picture
sucrose gradient for protein fractionation???

Hello,

I would like to have fractions of my whole cell extract, I'm trying to isolate nucleoplasmic and cytoplasmic fraction.
But my PI insists that I can use sucrose gradient for fractionation of my cell lysates.
I thought it is just for organelle fractionation and cannot be applied to my sample, which is whole cell lysate sup after spin.
Gradients kit contains from 8.5 to 60 percent sucrose.
Someone please help whether I can use the method. Thank you in advance!!

Chin Fen Teo
Chin Fen Teo's picture
 Hi minylim,

 Hi minylim,

Sucrose gradient is okay for isolate nucleoplasmic and cytoplasmic fraction, but there are many much easier methods to isolate these two fractions without going through the ultracentrifugation.... 

If you are looking for a kit to save your time in preparing buffers, I recommend this one from Pierce/Thermo Fisher.

Otherwise, you can perform the isolation with homemade buffers as follow:
Materials:
1. Hypotonic buffer- 5mM HEPES(pH7.9)/ 10mM KCl/ 1.5 mM MgCl2
2. 10% IGEPAL (or called NP-40) in ddwater
3. Hypertonic buffer- 5mM HEPES(pH7.9)/ 0.25% Np-40/ 25% glycerol/ 500mM NaCl/ 1.5 mM MgCl2/ 0.2mM EDTA

Procedures:
1. Resuspend cell pellet in 1 vol of ice-cold hypotonic buffer.
2. Add 0.02 vol of 10% NP-40 (final = 0.25% NP-40), vortex vigorously for ~ 10 sec.
3. Leave on ice for 10 min.
4. Centrifuge: 10k Xg, 4 deg, 30 sec. S/N= Cytoplasmic fraction, Pellet= Nuclei
5. Wash the nuclei with hypotonic buffer.
6. Resuspend Pellet with 0.2 vol of ice-cold hypertonic buffer. 
7. Incubate (end-to-end rotator) for 2 hours at 4 deg.
8. Centrifuge: 10k Xg, 4 deg, 10 min. S/N= Nuclear protein extract.

 Good luck.