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Nucleoprotein Extraction Protocol

Nucleoproteins are a class of binding proteins that structurally associated with nucleic acids, and widely exist in the nucleus of various biologic species. According to the type of nucleic acids, nucleoproteins can be divided into deoxyribonucleoproteins (DNA) and ribonucleoproteins (RNA). Studies focused on nucleoproteins are important for the understanding of disease-related genes and search of new proteomic markers for diseases. And the extraction of nucleoprotein is essential procedure for the analysis of specific spatial structure and biological functions. Here, we provide a protocol for nucleoprotein extraction from cultured cells and tissue in an efficient manner.

Reagents:

  • PBS: pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4.
  • Buffer A: pH 7.9, 10 mM HEPES, 1.5 mM MgCI2, 10 mM KCI, 1 mM DTT, with 1× protease inhibitor cocktail and phosphatase inhibitor cocktail.
  • Buffer B: pH 7.9, 20 mM HEPES, 1.5 mM MgCI2, 25% glycerol, 420 mM NaCI, 0.2 mM EDTA, 1 mM DTT, with 1× protease inhibitor cocktail and phosphatase inhibitor cocktail.
  • Buffer C: pH 7.9, 20 mM HEPES, 20% glycerol, 100 mM KCI, 0.2 mM EDTA, 1 mM DTT, with 1× protease inhibitor cocktail and phosphatase inhibitor cocktail.

Equipment:

  • Homogenizer

Steps:

Cultured cells preparation:

1. Centrifuge at 500 × g at 4°C for 5 min to harvest the cells

2. Wash the cells with cold PBS, centrifuge at 500 × g at 4°C for 2 to 3 min, and discard the supernatant.

3. Repeat Step 2, carefully discard the supernatant, leaving the cell pellet as dry as possible.

4. Add 1 mL cold Buffer A for 1 × 107 cells, vortex for 15 sec to fully suspend the cell pellet.

Skip to Step 10

Tissue preparation:

5. Weigh the tissue, cut the tissue into small pieces and then collect the fragment into a fresh 1.5 mL tube.

6. Wash the tissue with cold PBS, centrifuge at 500 × g at 4°C for 5 min

7. Discard the supernatant carefully to leave the pellet as dry as possible.

8. Add 1 mL cold Buffer A for 20~100 mg tissue, homogenize tissue using a homogenizer.

9. Vortex the tube vigorously for 15 sec to fully suspend the cell pellet.

Protein extraction:

10. Incubate the tube on ice for 10 min, and vortex again for 5 sec.

11. Centrifuge at 16,000 × g at 4 °C for 5 min, and immediately transfer the supernatant into a fresh 1.5 mL tube for cytoplasmic extraction.

12. Suspend the pellet with 0.75 mL cold Buffer B and vortex the tube for 15 sec.

13. Incubate on ice for 40 min, and vortex the tube for 15 sec very 10 min.

14. Centrifuge at 16,000 × g at 4 °C for 10 min, and immediately transfer the supernatant into a fresh 1.5 mL tube.

15. Reextract the residual pellet with 0.25 mL cold Buffer B as the same procedure describes at Step 14.

Dialysis (optional)

16. Following extraction, the nuclear extract is dialyzed for 2 hr against 100 volumes of Buffer C.

17. Centrifuge at 2000 × g at 4 °C for 10 min to remove precipitate.
Note: The short dialysis period results in less precipitation of denatured protein and is sufficient to adjust the extract to the desired ionic conditions.

18. Store at -80 °C until use.

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References:

1. Lee KA, et al., A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene Anal Tech. 1988, 5(2):22-31.
2. Zerivitz K, et al., An improved nuclear extract preparation method. Gene Anal Tech. 1989, 6(5):101-9.

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