Large scale nuclear extract preparation

I. Homogenize cells or tissue

1. Wash commercially available HeLa cells (30g) or minced in 3mm pieces fresh liver(15-20g) from staining with homogenizing buffer (10ml) and transfer it to 100ml beaker

2. Add to the beaker another 25ml of the buffer and split the content in half (2x15ml)

3. Homogenize each portion twice, 10 strokes (“up” and “down”) each with a teflon pestle. Between strokes, cool homogenizer on ice. Try to maintain temperature close to +4C

II. Pellet the nuclei

1. Filter homogenate through the four- layer cloth (25-30ml) and mix homogenate with cushion buffer (50ml of cushion buffer per 25ml of the homogenate

2. Distribute to ultracentrifuge tubes cushion buffer (10 ml of buffer per a tube). Carefully load cushion buffer- homogenate mix on the top (total volume is about 38 ml).

3 Equilibrate tubes at the balance and centrifuge them in SW28 rotor (24,000 rpm or 76220g, 50min, 1C) or 60Ti rotor (same time and temperature at 32,750 rpm/min)

III. Lyse nuclei

1. Invert tubes and keep them on ice for 10min

2. Resuspend pellets in nuclear lysis buffer (5ml/pellet). Perform one stroke with Dounce homogenizer

3 Add lysis buffer to 40ml and 2ml of 4M ammonium sulfate and agitate for 30-60min at +4C

IV. Reprecipitate nuclear proteins

1. Centrifuge lysates in 55.2Ti rotor (35,000rpm, 60min, +1C) or 60Ti rotor (37,700rpm, 60min, +1C).
Save super.

2. Add ammonium sulfate (330mg/ml of solid salt or 65ml of 4M solution) and keep sample(s) on ice for 15min

3 Centrifuge sample(s) in 55.2Ti rotor or 60Ti rotor for 20min at the same speed and temperature.
Save THE PELLET(S).

V. Dialyze nuclear extract and measure protein

1. Resuspend each pellet in 500ml of dialysis buffer and dialize sample(s) against dialysis buffer (1:1,000 for 4h)

2 Clarify nuclear extract by centrifugation (+4C, 10min) in microfuge.

3. Measure protein and aliquot supernatant (100-500mg/tube) and store it at -70C