Nuclear and cytoplasmic extraction

Nuclear and cytoplasmic extraction

The following was performed on the liver lysates: the cell pellet was suspended in a hypotonic buffer (cytoplasmic extraction buffer (CEB): 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1 mM EDTA, 0.05% NP40, pH 7.9). Detergent (NP40) was added and vortexed to separate the nuclei from the cytoplasmic fraction. The solution was centrifuged and the supernatant was collected (which contained the cytoplasm). The pellet (containing nuclei) was resuspended in a nuclear extraction buffer (nuclear extraction buffer (NEB): 5 mM HEPES, 1.5 mM MgCl2, 4.6 M NaCl, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol, pH 7.9) that busted the nuclear membrane. It was then centrifuged at high speed and the supernatant (nuclear fraction) was collected. The final pellet was discarded. TBP was used as a nuclear fractionation loading control.

Tissue Preparation 

1. Cut 20-100 mg of tissue into small pieces and place in a microcentrifuge tube. 

2. Wash tissue with PBS. Centrifuge tissue at 500 × g for 5 minutes. 

3. Using a pipette, carefully remove and discard the supernatant, leaving cell pellet as dry as possible. 

4. Homogenize tissue using a Dounce homogenizer or a tissue grinder in the appropriate volume of cell lysis buffer (CLB; Cell Signaling Tech; Cell Lysis Buffer (10X) #9803). 

Cytoplasmic and Nuclear Protein Extraction: Scale this protocol depending on the cell pellet volume. Maintain the volume ratio of Lysis CLB:CEB:NEB reagents at 200:11:100µL, respectively. 

1. Vortex the tube vigorously on the highest setting for 15 seconds to fully suspend the cell pellet. Incubate the tube on ice for 10 minutes. 2. Add ice-cold CEB to the tube. 

3. Vortex the tube for 5 seconds on the highest setting. Incubate tube on ice for 1 minute. 

4. Vortex the tube for 5 seconds on the highest setting. Centrifuge the tube for 5 minutes at maximum speed in a microcentrifuge (~16,000 × g). 

5. Immediately transfer the supernatant (cytoplasmic extract) to a clean pre-chilled tube. Place this tube on ice until use or storage (see Step 10).

6. Suspend the insoluble (pellet) fraction produced in Step 4, which contains nuclei, in ice-cold NEB. 

7. Vortex on the highest setting for 15 seconds. Place the sample on ice and continue vortexing for 15 seconds every 10 minutes, for a total of 40 minutes. 

8. Centrifuge the tube at maximum speed (~16,000 × g) in a microcentrifuge for 10 minutes. 

9. Immediately transfer the supernatant (nuclear extract) fraction to a clean pre-chilled tube. Place on ice. 

10. Store extracts at -80°C until use