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Finkelstein Lab Wiki

Rnaseh1 purification

RNase H1 purification, 1XFLAG-His. Size: Around 34 Kd.

Contents

Section titled “Contents”
  1. Expression
  2. Purification
  3. Buffers

1. Expression

Section titled “1. Expression”
  1. Grow 4 L at 37 C in presence of ampicillin or carbenicillin to OD .7/.8.
  2. Induce with .3 mM IPTG for 3 hr at 30C, Spin down cell pellets and freeze at -80 C.

2. Purification

Section titled “2. Purification”
  1. Resuspend cell pellets in Lysis buffer with protease inhibitor tablet and PMSF.
  2. Sonicate 30Amp, 30sec on 15sec off, 10 min.
  3. Spin down at 30,000 rpm for 1 hr.
  4. Load supernatant on Histrap FF Ni column (5 mL, preequilibrated with lysis buffer). Wash with Ni Wash Buffer. Elute with gradient of Elution buffer.
  5. Dilute protein 10X with (Heparin Dilution Buffer).
  6. Load on Hitrap Heparin HP (5ml, preequilibrated with Heparin Dilution Buffer). Wash with Heparin Wash Buffer. Elute with gradient of Heparin Elution Buffer.
  7. Dialyse against storage buffer or use pooled elutions (may be high salt), concentrate, store at -80.
h1_purification.png

3. Buffers

Section titled “3. Buffers”
  • Lysis Buffer: 25 mM Tris HCl pH 7.5, 10% glycerol, .1% Igepal, 10mM Imidazole, 0.5M NaCl
  • Ni Wash Buffer: 25 mM Tris HCl pH 7.5, 10% glycerol, .1% Igepal, 40mM Imidazole, 0.5M NaCl
  • Ni Elution Buffer: 25 mM Tris HCl pH 7.5, 10% glycerol, .1% Igepal, 350mM Imidazole, 0.5M NaCl
  • Heparin Dilution Buffer: 25 mM Tris HCl pH 7.5, 10% glycerol, 0.05% Igepal, 1mM DTT
  • Heparin Wash Buffer: 25 mM Tris HCl pH 7.5, 10% glycerol, 0.05% Igepal, 1mM DTT, 100 mM NaCl
  • Heparin Elution Buffer: 25 mM Tris HCl pH 7.5, 10% glycerol, 0.05% Igepal, 1mM DTT, 1M NaCl
  • Storage Buffer: 25 mM Tris HCl pH 7.5, 10% glycerol, 0.05% Igepal, 1mM DTT, 200 mM NaCl