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Isolation of Nucleolus-Enriched Fraction 1. Measure protein concentration of yeast nuclei by the Bradford method. Measure OD260 of nuclei
 

Summary: 90
Isolation of Nucleolus-Enriched Fraction
1. Measure protein concentration of yeast nuclei by the Bradford method. Measure OD260 of nuclei
after diluting 1 l of nuclei into 99 l of 1% SDS.
2. Dilute 10 mg of yeast nuclei into 5 volumes of PSM1 buffer + freshly added protease inhibitor
cocktails (PICs) + freshly added 1 mM DTT (from 1 M stock). Dilute nuclei in a 50-ml Oakridge
polycarbonate tube on ice. Vortex to completely disperse nuclei and to mix Ficoll with buffer.
3. Spin at 8000 rpm (~10,000 g max) for 10 minutes in Sorvall HB-4 rotor at 4C.
Begin setting up to pour the sucrose step gradients at this point.
4. Carefully remove all of the supernatant (SN) with an aspirator. The pellet will be a thin layer on
the bottom of the tube. Keep pellet on ice.
5. To 1 ml of PSM1 buffer on ice, freshly add PICs and 1 mM DTT. Resuspend the nuclear pellet in
cold PSM1 as follows: re-pipette for 1 minute, place on ice for 1 minute, vortex for 15 seconds,
add 5 l DNase I solution, and vortex again for 15 seconds.
Nuclei: 10 mg PSM1: 1 ml DNase I: 5 l (50 g)
DNase I is Sigma Type II (D4527). Prepare as 10 mg/ml stock in 50% glycerol, 20 mM KPi,
pH 7, 1 mM MgCl2, 1 mM DTT. Freeze as 10 l aliquots (100 g) at -70C.
6. Hold at room temperature for 4 minutes following resuspension.
7. Add 25 l (250 g) of heparin solution. Mix by gentle vortexing for 15 seconds.
Heparin is Sigma Grade I-A sodium salt (H-3393). Prepare as 10 mg/ml stock solution in

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine