p35_t

Growth

1. Transform BL21 DE3 [RIL] with DNA. Select on Amp plates.
2. Pick a single colony into 1 l 2xTY/Amp + ampicillin. Grow two nights and one day at 20°C with shaking (200 rpm).
3. Harvest by centrifugation at 5,000 rpm at 4°C for 10 minutes.
4. Resuspend in 5 ml sucrose buffer per litre of media. Store at -20°C.

Purification

Use PMSF throughout

1. Thaw pellets and lyse by sonication on ice (4 x 20s on 25% power with the medium tip should do it. Don't overcook).
2. <20k, 2°C, 20 min>
3. Dialyse at 4°C overnight versus 4l HiLo buffer.
4. <20k, 2°C, 20 min>
5. Load on to Q-Sepharose in HiLo buffer. Keep the flow-through. Elute with a 250 ml gradient to 500 mM NaCl (use HiHi as buffer B on the FPLC).
6. Concentrate the p35_t-containing fractions (about 100 mM NaCl) in Vivaspin 10s and apply to S-200 in Tris-Cl pH 8 + 50 mM NaCl + DTT, EGTA, PMSF.
7. Collect p35_t-containing fractions and concentrate/desalt in Vivaspin 10.
8. My first attempt at freezing was unsuccessful. For now, I keep the concentrated protein on ice in the cold room.

Quantification

Buffers