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)