Cell fractionation

Cell fractionation

Lab. Luciano Di Croce

1. Harvest 25 million cells by trypsinization, spin down at 150 x g for 5 minutes, and discard supernatant.

2. Wash cell pellet with PBS, spin down at 150 x g for 5 minutes, and discard supernatant.

3. Resuspend cell pellet in 10 volumes Buffer A by pipetting up/down (E.g. ≈ 50 µl pellet / 500 µl buffer). Collect an aliquot of 10% volume for Western Blot analysis, add Buffer B.SDS 2X (E.g. 50 µl cell extract + 50 µl buffer) and boil at 98ºC for 10 minutes -> Total Fraction (T).

4. Incubate the rest of the cell extract for 5 minutes on ice.

5. Centrifuge at 1,300 x g for 5 minutes at 4ºC to separate cytoplasmic (Supernatant 1) and nuclear (Pellet 1) frations.

6. Remove Supernatant 1, clarify by centrifugation (15,000 x g, 15 minutes, 4ºC) and transfer to a new tube. Collect an aliquot of 10% volume for Western Blot analysis, add Buffer B.SDS 2X (E.g. 50 µl clarified supernatant + 50 µl buffer) and boil at 98ºC for 10 minutes -> Cytoplasmic Fraction (C).

7. Wash Pellet 1 with 5 volumes Buffer A (E.g. 250 µl). Centrifuge at 1,300 x g for 5 minutes at 4ºC and discard supernatant. 

8. Resuspend the nuclear pellet in 1 volume Buffer A by gently pipetting up/down (E.g. 50 µl). Collect an aliquot of 10% volume for Western Blot analysis, add Buffer B.SDS 2X (E.g. 5 µl nuclear suspension + 95 µl buffer) and boil at 98ºC for 10 minutes -> Nuclear Fraction (N).

9. Add 10 volumes Buffer B (E.g. 500 µl). Do not pipette up/down! Incubate 30 minutes on ice and quick vortex.

10. Centrifuge at 1,700 x g for 5 minutes at 4ºC to separate the nucleoplasmic (Supernatant 2) and the insoluble chromatin (Pellet 2) fractions. 

11. Remove Supernatant 2, clarify by centrifugation (15,000 x g, 15 minutes, 4ºC) and transfer to a new tube. Collect an aliquot of 10% volume for Western Blot analysis, add Buffer B.SDS 2X (E.g. 50 µl clarified supernatant + 50 µl buffer) and boil at 98ºC for 10 minutes -> Nucleoplasmic Fraction (Np).

12.  Wash Pellet 2 with 5 volumes Buffer B (E.g. 250 µl). Do not pipet up/down. Centrifuge at 1,700 x g for 5 minutes at 4ºC and discard supernatant.

13. Resuspend pellet in 10 volumes Buffer B.SDS 1x (E.g. 500 µl) and boil at 98ºC for 10 minutes.

14. Sonicate until the sample is no longer viscous (in our hands, 4 cycles, each one 15sec ON/45sec OFF, in a Bioruptor  Diagenode) -> Chromatin Fraction (Chr).

For Western Blot, quantify proteins in the Total Fraction by BCA assay, and analyze protein samples by SDS polyacrylamide-gel electrophoresis. Run the amount of Total Fraction required to detect your target protein (between 5-100 µg, according to the abundance of the protien and antibody sensitivity), and run the same volume of all the fractions (T, C, N, Np, Chr). For fractionation markers as GAPDH (T, C); PCNA (T, N, Np, very little on Chr), H3 (T, N, Chr), 5ug of total protein are enough.

Buffer A: 

10 mM HEPES pH 7.9; 10 mM KCl; 1.5 mM MgCl₂; 0.34 M Sucrose; 10% Glycerol; 1mM DTT; 0.1 % Triton x100. Add protease / phosphatase inhibitors immediately before use.

Buffer B: 

3 mM EDTA pH 8.0; 0.2 mM EGTA pH 8.0; 1 mM DTT. Add protease / phosphatase inhibitors immediately before use.

Buffer B.SDS 2X: 

25 mM TrisCl pH 7.5; 1% SDS; 1 mM EDTA pH 8.0

Protease / phosphatase inhibitors: 

1x Complete EDTA-free protease inhibitor cocktail (Roche); 1 mM PMSF; 1 mM Na₃VO₄; 10 mM β-Glycerophosphate