Fractionation and Extraction of Crude Nuclear Proteins From Arabidopsis Seedlings

Materials and Reagents

1. Materials

   1. 200 μL pipette tips

   2. 1 mL pipette tips

   3. 5 mL pipette tips

   4. 100-μm Nylon cell stainer (Corning Incorporated, FALCON, catalog number: 352350)

   5. 40-μm Nylon cell stainer (Corning Incorporated, FALCON, catalog number: 352340)

   6. 50 mL centrifuge tube

   7. 1.5 mL centrifuge tube

   8. Arabidopsis seedlings

   9. Liquid nitrogen

      
      

2. Reagents

   1. Sucrose (Sinopharm, SCR, catalog number: 10021418)

   2. Ficoll 400 (Sigma-Aldrich, catalog number: F9378-5G)

   3. Dextran T40 (Solarbio, catalog number: D8250)

   4. Tris (Sangon Biotech, catalog number: A610195)

   5. HCl (Sinopharm, SCR, catalog number: 10011018)

   6. MgCl₂ (RHAWN, catalog number: R007504-500 g)

   7. Triton X-100 (Sigma-Aldrich, catalog number: V900502-100ML)

   8. phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: PMSF-RO)

   9. β-mercaptoethanol (Sigma-Aldrich, catalog number: 07604-100ML)

   10. Protease inhibitor cocktail (Roche, cOmplete Tablets EDTA-free, EASYpack, catalog number: 04693132001)

   11. KCl (Sinopharm, SCR, catalog number: 10016318)

   12. HEPES (Sigma-Aldrich, catalog number: H3375)

   13. Hexylene glycol (Sangon Biotech, catalog number: A607060)

   14. Spermine (Sigma-Aldrich, catalog number: S3256)

   15. Spermidine (Sigma-Aldrich, catalog number: S2626)

   16. Glycerol (Sangon Biotech, catalog number: A100854)

   17. NaCl (Sinopharm, SCR, catalog number: 10019318)

   18. 0.5 M EDTA, pH 8.0 (Biosharp, catalog number: 69075458)

   19. Dithiothreitol (DTT) (Beyotime, catalog number: ST043-5g)

   20. Urea (Sangon Biotech, catalog number: A600148)

   21. Nonidet P-40 (NP-40) (Roche, catalog number: 11332473001)

   22. Anti-Histone H3 antibody (Abcam, catalog number: ab18521)

       
       

3. Stock solution

   Stock solutions for the buffers can be made before and stored at 4°C (1-5) or -20°C (6-10). On the day of the experiment, the buffers can be diluted as required.

   1. 1 M Tris-HCl, pH 7.4

   2. 20% Triton X-100 (v/v)

   3. 20% NP-40 (v/v)

   4. 2 M KCl

   5. 0.5 M HEPES-KOH, pH 7.0

   6. 50 mM Spermine

   7. 125 mM Spermidine

   8. 10 mM PMSF

   9. 1.0 M DTT

   10. Protease inhibitor cocktail (100×) (see Recipes)

   11. Honda buffer (see Recipes)

   12. Nuclei isolation buffer (NIB) buffer (see Recipes)

   13. Glycerol buffer (see Recipes)

   14. Nuclei lysis buffer (see Recipes)

Equipment

1. Centrifuge (ThermoFisher, catalog number: 75004380)

2. Vortex shaker

3. Mortar

Procedure

1. Nuclei isolation

   1. Transfer 2.0 g of the 10-day-old Arabidopsis seedlings into a mortar, which is prechilled with liquid nitrogen. Grind in liquid nitrogen for about 5 min until a fine powder is obtained. Transfer the frozen seedling powder into a 50 mL tube filled with 4 mL of precooled Honda buffer (2 mL/g), mix by vortex, then incubate on ice for 10 min.

   2. Filter the homogenate through a 100-μm and then a 40-μm mesh nylon cell stainer sequentially, take 100 μL of the flow-through as the total protein extract (control 1), then centrifuge the remaining flow-through at 1,500 × g for 5 min at 4°C.

   3. Transfer 100 μL of the supernatant to a new tube, then centrifuge at 12,000 × g for 10 min at 4°C, collect the supernatant as the cytosol fraction (control 2).

   4. Discard the remaining supernatant, wash the dark-green pellet containing a crude nuclear fraction with 5 mL of nuclei isolation buffer (NIB). Gently pipet up and down with a 5 mL-sterile pipette. Centrifuge at 1,500 × g for 10 min at 4°C, discard the supernatant, and pellet the nuclei.

   5. Repeat Step A4 three or four times, until the color of the nuclear pellet becomes white, with as little green coloration as possible. The white nuclear pellet is regard as the crude nuclei fraction.

      
      

2. Nuclear protein extraction

   1. Gently resuspend the nuclear pellet in 30 μL of prechilled glycerol buffer and 30 μL of nuclei lysis buffer, then mix the nuclei pellet by vortexing, and incubate on ice for 10 min, to obtain the lysis of the nucleus as the nuclear fraction.

   2. Add 12 μL of 6× SDS protein loading buffer and mix with the nucleus fraction by vortexing, then boil at 100°C for 10 min. Centrifuge at 12,000 × g for 10 min at room temperature. Aspirate the supernatant (nuclear proteins) to a new tube, store the sample at -80°C untill use.

Data analysis

1. Before nuclear protein extraction, check the quality of nuclei through 4',6-diamidino-2-phenylindole (DAPI) (0.33 μg/mL) staining of the crude nuclei, and estimate the ratio of intact nuclei under a fluorescent microscope immediately after staining (Figure 1A).

2. Estimate the quality of nuclear proteins by western blot using anti-Histone H3 antibody (Abcam, USA, catalog number: ab18521), using the total protein extract (control 1) and the cytosol fraction (control 2) as controls. Histone H3 is found in the total protein and nucleus fractions, but not in the cytosol fraction, suggesting that the nucleus fraction is enriched in nuclear proteins (Figure 1B).

   
   

   
   Figure 1. Estimation of the quality of nuclear proteins. (A) Observation of nuclei (white points) under a fluorescent microscope after DAPI staining. (B) Western blot analysis using anti-Histone H3 antibody. The total protein extract (total), the nuclear fraction (nucleus), and the fraction devoid of nucleus (cytosol).

Notes

1. All the steps for isolation of nuclei are performed on ice or at 4°C.

2. Add protease inhibitors (e.g., PMSF and protease inhibitor cocktail) to extraction solutions at the moment before you start.

Recipes

1. Protease inhibitor cocktail (100×)

   Dissolve one tablet in 500 μL of ddH₂O.

2. Honda buffer

   0.4 M Sucrose

   2.5% Ficoll

   5% Dextran T40

   25 mM Tris-HCl, pH 7.4

   10 mM MgCl₂

   0.5% Triton X-100

   0.5 mM phenylmethylsulfonyl fluoride (PMSF)

   10 mM β-mercaptoethanol

   1× Plant protease inhibitor cocktail

3. Nuclei isolation buffer (NIB) buffer

   20 mM KCl

   20 mM HEPES, pH 7.4

   0.5% Triton X-100

   13.8% hexylene glycol

   0.1% (v/v) β-mercaptoethanol

   50 μM spermine

   125 μM spermidine

   1 mM PMSF

   1× Plant protease inhibitor cocktail

4. Glycerol buffer

   20 mM Tris-HCl, pH 7.9

   50% (v/v) glycerol

   75 mM NaCl

   0.5 mM EDTA

   0.85 mM Dithiothreitol (DTT)

   0.125 mM PMSF

   1× Plant protease inhibitor cocktail

5. Nuclei lysis buffer

   10 mM HEPES, pH 7.6

   1 mM DTT

   7.5 mM MgCl₂

   0.2 mM EDTA

   0.3 M NaCl

   1 M Urea

   1% Nonidet P-40 (NP-40)

   0.5 mM PMSF

   1× Plant protease inhibitor cocktail

Acknowledgments

This protocol was adapted from the methods described in Liu et al. (2018) and Calikowski and Meier (2006).

Competing interests

The authors declare no conflicts of interest.

References

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2. Komatsu, S. (2007). Extraction of Nuclear Proteins. In: Plant Proteomics: Methods and Protocols. Thiellement, H., Zivy, M., Damerval, C., and Méchin, V. (Eds.). Methods in Molecular Biology. Humana Press: Totowa, NJ. pp. 73-77.
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