p15

Growth

1. Transform BL21 DE3 [RIL] with pET9-p15 DNA.

2. Option A

   1. Pick a single colony into 50 ml 2xTY with Cam/Kan. Grow overnight at 37 °C.
   2. Add 50 ml to 1 l 2xTY/Kan. Grow at 20°C until A₆₀₀ = 0.6.

   Option B

   1. Pick a single colony into 1 l 2xTY/Kan/Cam. Grow for 30 - 36 hr at 20°C until A₆₀₀ = 0.6.

3. Add IPTG to 0.2 mM and grow for a further 2 hours at 20°C.
4. Harvest by centrifugation at 5,000 rpm at 4°C for 10 minutes.
5. Resuspend in 10 ml sucrose buffer per litre of media. Store at -20°C.

Purification

1. Thaw pellets and lyse by sonication on ice (3 x 20s on 20% power with the medium tip should do it. Don't overcook).
2. Dilute 1 in 2 with Buffer A and add RNase to 10 µg ml^-1.

3. Centrifuge at 20,000 rpm at 2°C for 15 minutes.

4. Chill supernatant on ice.
   1. Add 16.4 g ammonium sulphate per 100 ml (= 30%). Mix until dissolved and leave on ice, 30 minutes.
   2. Spin 20k, 2°C, 10 min.
   3. To the supernatant add 14.8 g AmS per 100 ml (= 55%). Mix until dissolved and incubate on ice for 30 minutes, with occasional mixing.
   4. Spin 20k, 2°C, 10 min. The p15 is in the pellet.
   5. (Not really necessary - add AmS at 27.3 g/100 ml to bring up to 95% and pellet everything else.)

5. Resuspend the 55% AmS pellet in 50 - 100 ml Buffer A and dialyse vs 1 l NTF Buffer overnight.

6. Spin 20k, 2°C, 10 min.

7. Apply to Q-Seph column in NTF Buffer and elute with a gradient to 1 M NaCl over 200 ml. p15 elutes around 0.1 M NaCl.

8. Pool p15-containing fractions, concentrate in CentriPrep 3 and separate over a S-100 (or similar) in p15 Buffer.

Quantification

Buffers