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.

Mw
mM
Add per L
HEPES (pH 7.4)
238.30
20
4.77 g
KCl
75.55
10
0.75 g
MgCl2
95.21
2
0.19 g
EDTA
292.24
1
0.29 g
EGTA
380.35
1
0.38 g
Stocks
Per 10 mL
1 mM DTT
1 M
10 µL
PI Cocktail (III)
-
50 µL

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.

Wash nuclear pellet remaining after Step 4 with 500 μL of fractionation buffer.

Resuspend the pellet in TBS with 0.1% SDS.