Histone Extraction Protocol

 

1. Cell Preparation

1.1 Centrifuge suspended cells at 300×g for 5 min at 4°C. Carefully remove the supernant, leaving the precipitate.

1.2 Add 2~3ml 1×PBS to the pellet and vortex.

1.3 Centrifuge suspended cells at 300×g for 5 min at 4°C. Carefully remove the supernant, leaving the precipitate.

1.4 Add 2~3ml 1×PBS to the pellet and vortex. 

1.5 Centrifuge at 300×g for 5 min at ℃. Aspirate the supernatant, save pellet.

 

2. Extraction

2.1 Add 2X the precipitation volume of pre-cooled buffer A (approximately 300 uL Buffer A per 1.0×107 cells), and gently pipette to suspend the pellet.

2.2 Incubate on ice for 15 minutes.

2.3 Add 1X volume of 10% NP-40 solution (300 uL Buffer A and 30 uL NP-40 solution), and vortex for 10 seconds.

2.4 Centrifuge at 10,000×g for 20 seconds at 4°C.

2.5 Collect the supernatant, which is the theoretical cytoplasmic protein, and transfer it to a clean centrifuge tube, which can be used immediately or frozen (stored at -20°C).

2.6 Completely aspirate the remaining supernatant in the pellet and discard it, and then add 1 times the precipitation volume of pre-cooled Buffer B (approximately 50 uL of buffer for 1.0×107 cells) and gently pipette to suspend the precipitate.

2.7 Place on a shaker at 4℃ and shake at the lowest speed for 30 min.

2.8 Centrifuge at 16,000×g for 5 min at 4°C, collect the supernatant as the nuclear protein lysate, transfer to a clean centrifuge tube, store at -20°C, and quantify the protein concentration by BCA.

 

Suggested Buffers:

Buffer A

  • 10 mmol/L HEPES-NaOH (pH7.9)
  • 15 mmol/L KCl
  • 1 mmol/L MgCl2
  • 0.1 mmol/L EDTA
  • 1 mmol/L DTT,
  • 1 mmol/L PMSF (add at time of use)
  • 1 mmol/L 100× protease inhibitor (add at time of use)

Buffer B

  • 20 ​​mmol/L HEPES-NaOH (pH7.9)

  • 0.42 mol/L NaCl

  • 1.5 mmol/L MgCl2

  • 0.2 mmol/L EDTA

  • 25% glycerol

  • 0.5 mmol/L DTT

  • 0.5 mmol/L PMSF (add at time of use)

  • 1 mmol/L 100× protease inhibitor (add at time of use)

 

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