Purification of His-tagged proteins - denaturing conditions


    Expression and isolation of inclusion bodies

  • transform E. coli BL21 with the expression plasmid and inocculate an overnight culture (100 ml)
  • induce expression in 500 ml LB+Amp under the optimised conditions by IPTG (OD600, induction time, temperature). Take 1 ml samples at all stages of the procedure to control the expression. Suspend the samples to a density of 50 ul Lammli for 1ml of OD600.
  • collect the cells by centrifugation (10 min, 5k)
  • suspend in 25 ml of lysis butter with a glass pipette. Add a tip of a spatula of DNAse. You may freeze the suspension at this stage.
  • Lyse the cells with the microfluidizer in Ficner's lab. Please consult an experienced person, if you are not familiar with the maschine!
  • Spin for 20 at 5k.

    • Solubilisation and purification with Ni beads

  • Suspend the pellet thoroughly in 25 ml of buffer A with a glass pipette. Start with a small volume, then dilute with remaining volume.
  • Stir suspension with a stir bar slowly for 30-60 min at RT
  • Spin at 10k for 10min, if necessary repeat centrifugation. The pellet is often not stable.
  • Equilibrate 3 ml Ni beads with puffer A, let beads settle down, remove carefully the supernatant. Be carefull not to loose any of the beads
  • Add the solubilised pellet extract to the beads
  • incubate for 30-60 min at RT, gently mix on a wheel
  • Prepare a drop column with puffer A
  • Fill the suspension into column, collect and save the flow-through
  • Wash the beads with 3 x 6ml with buffer C (at least 3 column volumes). Be careful when adding buffer to the beads
  • Elute protein with buffer E, add volumes of 1 ml (0.5 column volumes). Collect the eluate in separate tubes. The protein will start to elute at 1-2 volumes. Do not suspend the beads when adding the elution buffer
  • Collect in total about 15 fractions,

    • analysis:

  • spot 1 ul of each fraction onto a stripe of nitrocellulose. You may use the same pipette tip. Stain with amidoblack.
  • Analyse the samples of the procedure together with sample of the elution fraction by SDS-PAGE. Samples containing guanidinium - puffer A can not be used, since a precipitate forms when lŠmmli is added.

solutions, buffer:

Since the urea is not stable even at 4¡C during storage, the ph has to be adjusted at the same day. Urea buffers are stored at 4C, where they will form crystalls. The buffer A may be stored at room temperature.

lysis buffer:
(20 mM Na-Phosphate pH 8, 500 mM NaCl, 20 mM imidazol
buffer A:
0,1M Na-Phosphate, 10 mM Tris pH 8 (NaOH), 6M GuHCl

1,38 g NaH2PO4xH2O
0,12 g Tris base
57,3 g guanidine hydrochloride
dH2O ad 100 ml, filter, store at RT
buffer C:
0,1 M Na-Phosphate, 10 mM Tris pH 6,3 (HCl), 8 M urea
1,38 g NaH2PO4xH2O
0,12 g Tris base
48,05 g urea
dH2O ad 100 ml
store at 4°C, always readjust the pH
buffer E:
0,1 M Na-Phosphate, 10 mM Tris pH 4,5 (HCl)
1,38 g NaH2PO4xH2O
0,12 g Tris base
48,05 g urea
dH2O ad 100 ml
store at 4°C, always adjust the pH prior to use