Nuclear extraction and fractionation
Procedure for extracting and fractionating the nuclear fraction of cells using centrifugation methods.
Last edited Sun 28 Aug 2022
Convenient kits for nuclear extraction and fractionation
For reliable results when extracting the nuclear fraction, we recommend our nuclear extraction kit (ab219177). Alternatively, we use the protocol below.
Stage 1 - Procedure
All centrifugation should be done at 4°C. Samples should be kept on ice throughout the procedure.
Steps
Transfer cells from 10 cm plates into 500 μL fractionation buffer, eg by scraping. Incubate for 15 min on ice.
- Just before use, add the following per 10 mL:
Using 1 mL syringe pass cells suspension through a 27 gauge needle 10 times (or until all cells are lysed).
Leave on ice for 20 mins.
Centrifuge sample at 720 x g for 5 minutes.
- The pellet will contain nuclei, and the supernatant will contain cytoplasm, membrane, and mitochondria.
Wash nuclear pellet remaining after Step 4 with 500 μL of fractionation buffer.
- Disperse the pellet with a pipette and pass through a 25 gauge needle 10 times.
- Centrifuge again at 720 x g for 10 minutes.
- Discard the supernatant and keep the pellet containing nuclei.
Resuspend the pellet in TBS with 0.1% SDS.
- Sonicate the suspension briefly to shear genomic DNA and homogenize the lysate (3 sec on ice at a power setting of 2-continuous).