FxFG 5- & 18-mer repeats

Growth

  1. Transform BL21 DE3 with RSA16 (FF5) or RD26 (FF18) DNA.
  2. Pick single colonies into 1l 2xTY/amp. Grow overnight at at 34°C, 180 rpm 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

Important Note

You MUST use lots of PMSF throughout.

  1. Thaw pellets and lyse by sonication.
  2. <20k, 2°C, 20 min>
  3. Dialyse the supernatant overnight vs NSP-1
  4. <20k, 2°C, 20 min>
  5. Use the S-Seph FPLC column and program 3, bank 1 (Buffer A = NSP-1, Buffer B = NSP-1+1 M NaCl. Gradient to 150 mM NaCl over 200 ml). Collect 5 ml fractions; FF18 comes off in fractions 11 - 16.
  6. Concentrate the FxFG-containing fractions in a Vivaspin 20, 10 kDa cutoff, and load onto the S-200 column on the FPLC in NSP-2. MAKE SURE THE COLUMN IS WELL WASHED WITH NSP-2 - imidazole interferes with spectro analysis. Use program 1, bank 1 and collect 7.5 ml fractions.
  7. Collect fractions 11 - 15 and concentrate pure FxFG-repeats in a Vivaspin. Freeze on liquid N2 and store at -20 or -80 °C.

Quantification

Coefficient at 280 nm = M-1 cm-1.

Mr = .

Buffers

NSP-1 NSP-2
20 mM imidazole pH 6.5
1 mM EGTA
1 mM DTT
0.1 mM PMSF
  		20mM Tris pH 7.0
50mM NaCl
0.2 mM EGTA
0.2 mM DTT
0.1 mM PMSF
RPG 2000/06/14, rev 2003/02/12

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