TapC2 - Y47D

TapC2 was made by PCR from human Tap1, residues 550 (methionine) to 619 (C-terminus). Cloned into pMW172 NdeI/EcoRI.

Growth

  1. Transform BL21 DE3 [RIL] with pMW172/TapC2 DNA. Select on Amp plates.
  2. Pick a single colony into 1 l 2xTY/Amp. Grow overnight at 37°C with shaking.
  3. Harvest by centrifugation at 5,000 rpm at 4°C for 10 minutes.
  4. Resuspend in 10 ml sucrose buffer per litre of media. Store at -20°C.

Purification

  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 TapC2 with a 100 ml gradient to HiHi.
  6. Concentrate the TapC2-containing fractions in Centriprep-3 (or Vivaspin 5) and apply to S-100 in HiLo buffer + 100 mM NaCl.
  7. Collect TapC2-containing fractions and concentrate (Centriprep/con 3/V5).

Quantification

Coefficient at 280 nm = 11,380 M-1 cm-1.

Mr = 7,843.

Enter
A280 reading 		Enter
dilution factor 		  Conc (mM) Conc (mg ml-1)

Buffers

HiLo HiHi
20 mM Tris-Cl pH 8.5
1 mM EGTA
1 mM DTT
 		 20 mM Tris-Cl pH 8.5
1 mM EGTA
1 mM DTT
500 mM NaCl
RPG 2000/07/03

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