Materials and Reagents

Note: All stock solutions should be stored at room temperature except when indicated otherwise.

Microcentrifuge tubes (Sigma-Aldrich, catalog number: Z336769 )
10 cm tissue culture dishes (Thermo Fisher Scientific, Nunc^TM, catalog number: 150350 )
Cell scraper (TPP Techno Plastic Products, catalog number: 99002 )
Pipette tips (STARLAB, TipOne^® filter tips, catalog number: S1120 )
Neural stem cells Cor3-1 (from Steve Pollard, University of Edinburgh)
P19 cells (ATCC, catalog number: CRL-1825 )
Protease cocktail inhibitor (Roche Diagnostics, catalog number: 04693116001 )
500 mM HEPES-KOH, pH 7.5 prepared from powder (Sigma-Aldrich, catalog number: H3375 )
0.5 mM EDTA, pH 8.0 (Sigma-Aldrich, catalog number: 93283 )
500 mM EGTA, pH 8.0 prepared from powder (Sigma-Aldrich, catalog number: E3889 )
5 M NaCl prepared from powder (Sigma-Aldrich, catalog number: S7653 )
1 M MgCl₂ prepared from powder (Sigma-Aldrich, catalog number: M8266 )
1 M Tris-HCl, pH 8.0 prepared from powder (Sigma-Aldrich, catalog number: RDD008 )
1 M Tris-HCl, pH 7.5 prepared from powder (Sigma-Aldrich, catalog number: RDD008 )
1 M Tris-HCl, pH 6.8 prepared from powder (Sigma-Aldrich, catalog number: RDD008 )
50% Glycerol (Sigma-Aldrich, catalog number: G5516 ) diluted in deionized water
10% SDS (Sigma-Aldrich, catalog number: 71736 )
10% NP-40 (Sigma-Aldrich, catalog number: I8896 ) diluted in deionized water
10% Triton X-100 (Sigma-Aldrich, catalog number: T8787 ) diluted in deionized water
100 mM DTT prepared from powder (Sigma-Aldrich, catalog number: 43815 ), store at -20 °C
PBS (Thermo Fisher Scientific, catalog number: 10010023 )
Benzonase 10 ku (Merck, Novagen^®, catalog number: 70664-3 )
α-Tubulin antibody (Abcam, catalog number: ab7291 )
LaminB (Santa Cruz Biotechnology, catalog number: sc-6216 or Gene Tex, catalog number: GTX103292 )
Histone H3 (Abcam, catalog number: ab1791 )
E1 Buffer (see Recipes)
E2 Buffer (see Recipes)
E3 Buffer (see Recipes)
E3 (benzonase) Buffer (see Recipes)

Equipment

Pipettes
Nanodrop (e.g., Thermo Fisher Scientific, model: NanoDrop^TM 3300 )
Water bath sonicator (e.g., Diagenode, model: Bioruptor^® )
Refrigerated centrifuge (e.g., Eppendorf, model: 5415 R )
Rotating platform (e.g., Grant Instruments, 360° Vertical Multi-function Rotator)

Procedure

Notes:

As guidance for cell density, neural stem cells Cor3-1 were seeded at 1.5 x 10⁶ in 10 cm dishes and P19 cells were seeded at 1.0 x 10⁶ in 10 cm dishes to reach 70%-80% confluency.
Before starting, make sure the centrifuge is chilled at 4 °C.

Wash cells once with room temperature PBS.
Collect cells with a cell scraper in 1 ml ice-cold PBS and transfer into a 1.5 ml microcentrifuge tube.
Pellet cells at 130 x g in a refrigerated centrifuge at 4 °C for 3 min.
Lyse cell pellet with 5 volumes of ice-cold E1 buffer for 1 volume of cell pellet.
Pipet gently up and down (5 times) before centrifugation at 1,100 x g at 4 °C for 2 min.
Collect the supernatant in a fresh tube (cytoplasm fraction).
Resuspend the pellet by gentle pipetting with the same volume of E1 buffer used for the previous step (Step 4).
Centrifuge immediately at 1,100 x g at 4 °C for 2 min.
Discard the supernatant and resuspend the pellet by gentle pipetting with the same volume of E1 buffer used for the previous step (Step 4).
Leave the tube for 10 min on ice.
Centrifuge at 1,100 x g at 4 °C for 2 min.
Discard the supernatant.
Resuspend the pellet by gentle pipetting in 2 volumes of ice-cold E2 buffer for 1 volume of cell pellet.
Centrifuge at 1,100 x g at 4 °C for 2 min.
Collect the supernatant in a fresh tube (nucleus fraction).
Resuspend the pellet with the same volume of E2 buffer used for the previous step (Step 13).
Centrifuge immediately at 1,100 x g at 4 °C for 2 min.
Note: The pellet should now start to be more difficult to dissolve by pipetting; using wide bore tips might be required.
Resuspend the pellet keeping the same volume of E2 used for the previous step (Step 13).
Leave the tube for 10 min on ice.
Centrifuge at 1,100 x g at 4 °C for 2 min.
Discard the supernatant.

For Western blot analysis:

Resuspend the pellet by pipetting in ice-cold E3 buffer (same volume as used for E1 in Step 4).
Note: The pellet is very sticky at this stage and precaution should be taken not to lose it.
Sonicate in a water bath sonicator with ice for 5 min, 30 sec ON/30 sec OFF on maximum power (i.e., setting H on Bioruptor).
Note: Ice should be replenished if necessary to keep a constant layer of crushed ice on top of the water bath.
Centrifuge all the fractions (i.e., cytoplasm, nuclear and chromatin) at 16,000 x g at 4 °C for 10 min.
Transfer each supernatant into fresh microcentrifuge tubes.
Measure protein concentration (e.g., Pierce BCA Protein Assay kit, Thermo Fisher Scientific, using a NanoDrop) and proceed to Western blot analysis.
Note: The protein concentration of the nuclear fraction might be low, and it could be necessary to precipitate proteins using, for example, Trichloroacetic acid method (see Paredes-Osses and Hardie, 2013).

For pull-down assays:

Resuspend the pellet in ice-cold E3 (benzonase) buffer (same volume as used for E1 in Step 4).
Flick the tube to detach the pellet.
Add benzonase at a 1:1,000 dilution.
Place the tube on a rotating platform and leave to rotate at 15 rpm for 20 min at room temperature.
Note: Incubation up to 30 min might be required to digest nucleic acids. NP-40 can be replaced with 0.1% SDS to enhance the reaction but the presence of SDS may interfere with downstream applications.
Centrifuge all the fractions at 16,000 x g at 4 °C for 10 min.
Transfer each supernatant into fresh microcentrifuge tubes.
Measure protein concentration and proceed to required pull-down assay (e.g., immuno-precipitation or resin pull-down).

Data analysis

Representative data for the efficiency of the cellular fractionation and for ubiquitin pull-down assay are shown in Gillotin et al. (2018) (https://www.nature.com/articles/s41598-018-23056-4.pdf). To assess the efficiency of the cellular fractionation, it is recommended to analyze each fraction by Western blot analysis for α-Tubulin, LaminB and Histone H3 as representative of the cytoplasmic, nuclear and chromatin fractions respectively.

Recipes

Note: Prepare all the solutions fresh from stock solutions before starting and keep on ice. Each solution is complemented with protease inhibitor cocktail and adding specific inhibitors should be considered to study post-translational modifications (e.g., phosphatase inhibitor, DUBs inhibitor).

E1 buffer
50 mM HEPES-KOH (from 500 mM stock solution, pH 7.5)
140 mM NaCl (from 5 M stock solution)
1 mM EDTA (from 500 mM stock solution, pH 8.0)
10% glycerol (from 50% stock solution)
0.5% NP-40 (from 10% fresh solution)
0.25% Triton X-100 (from 10% fresh solution)
1 mM DTT (from 100 mM stock solution, kept at -20 °C)
Supplemented with 1x protease inhibitor cocktail
E2 buffer
10 mM Tris-HCl (from 1 M stock solution, pH 8.0)
200 mM NaCl (from 5 M stock solution)
1 mM EDTA (from 500 mM stock solution, pH 8.0)
0.5 mM EGTA (from 500 mM stock solution, pH 8.0)
Supplemented with 1x protease inhibitor cocktail
E3 buffer
500 mM Tris-HCl (from 1 M stock solution, pH 6.8)
500 mM NaCl (from 5 M stock solution)
Supplemented with 1x protease inhibitor cocktail
E3 (benzonase) buffer
50 mM Tris-HCl (from 1 M stock solution, pH 7.5)
20 mM NaCl (from 5 M stock solution)
1 mM MgCl₂ (from 1 M stock solution)
1% NP-40
Supplemented with 1x protease inhibitor cocktail
Add Benzonase when ready to use (1:1,000 dilution)

Acknowledgments

Illustrations have been modified from the Servier Medical Art service (Les Laboratoires Servier). This work was adapted in Anna Philpott’s laboratory (University of Cambridge) from protocols established in Jason Carroll's laboratory (University of Cambridge). The author is currently supported by funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 713140 (The Meso_Brain Project, www.mesobrain.eu).

Competing interests

The author declares no competing interest.

References

Dennis, D. J., Han, S. and Schuurmans, C. (2018). bHLH transcription factors in neural development, disease, and reprogramming. Brain Res S0006-8993(18)30148-3.
Gillotin, S., Davies, J. D. and Philpott, A. (2018). Subcellular localisation modulates ubiquitylation and degradation of Ascl1. Sci Rep 8(1): 4625.
Gillotin, S. and Guillemot, F. (2016). Micro-chromatin immunoprecipation (μChIP) protocol for Real-time PCR analysis of a limited amount of cells. Bio-protocol 6(12): e1846.
Paredes-Osses, E. and Hardie, K. R. (2013). Cell Fractionation of Pseudomonas aeruginosa. Bio-protocol 3(19): e922.