IHF Purification Protocol

Day 1

  1. Streak out G509 (IHF_, IHF_ operon in pRSETb, via pRSUB, derivative) from frozen glycerol stock onto fresh LB+agar+Amp100 plate. 37° C overnight.

Day 2

  1. Innoculate 2 x 5 ml LB+Amp100 cultures. 37° C overnight.

Day 3

  1. Perform standard alkaline lysis phenol/chloroform plasmid prep
  2. Dialyze DNA against nanopure water for 30 minutes
  3. Transform into electrocompetent BL21 cells (contains pLysS plasmid)
  4. Plate on LB+agar+Amp100/Cam30. 37° C overnight.

Day 4

  1. Pick a colony and inoculate 100 ml of LB+Amp100/Cam30

Day 5

  1. 1. Subculture 1:100 in LB+Amp100/Cam30
  2. 2. 6 x 1 liter in 2.8 liter flasks, 250 rpm, 37° C
  3. Grow until OD600 = 0.5 (this took 4.5 hours)
  4. Induce with IPTG to final of 0.5 mM (0.5 ml of 1 M)
  5. Grow at 37° C for 3 hours (OD600 = 1)
  6. Transfer to 500 ml centrifuge bottles
  7. Centrifuge at 8,000 rpm for 15 minutes, decant supernatant
  8. Store pellets at -80° C (15 g of cells)

Day 6

  1. Remove bottles from freezer and combine frozen pellets
  2. Weigh cells (15 g of cells)
  3. Add 48 ml of buffer A:
      Stock for 50 mL
    50 mM Tris-HCl pH 8.0 1 M 2.5 ml
    10% Sucrose   5 g
    12.5 mM EDTA 0.5 M 1.25 ml
    2.5 mM DTT 1 M 125 ml
    Water 1 M Up to 50 ml

    Buffers made fresh before use
  4. Use 30 ml homogenizer (mortar and pestle type) to resuspend cells in buffer
  5. Add protease inhibitors:
    • 450 mg leupeptin
    • 450 mg pepstatin A
    • 900 mg soybean trypsin inhibitor
    • 45 mg PMSF (in 1 ml EtOH)
  6. Add lysozyme (1/20th volume @ 10 mg/ml in 0.25 M Tris-HCl pH 8.0)
  7. Homogenize several times to mix up protease inhibitors and lysozyme
  8. Add 12 ml buffer B:
      Stock for 15 mL
    200 mM Tris-HCl pH 8.0 1 M 3 ml
    1 M NaCl   0.875 g
    200 mM spermidine   0.775 g
    Water   Up to 15 ml

  9. Add 20 ml Benzonase nuclease (Novagen)
  10. Rotate in coldroom (4° C) for 2 hours
  11. Place in 37° C shaker for 5 minutes
  12. Split into 2 centrifuge bottles
  13. Spin in Ti55.2 rotor at 40,000 rpm for 1 hour
  14. Remove supernatant, add same amount of protease inhibitors from above and keep on ice
  15. Re-extract pellet in 8 ml buffer A and 2 ml buffer B and proceed as above
  16. Combine both supernatant and filter through steritop filter
  17. Freeze clarified extract in liquid nitrogen

Day 7

  1. Prepare 1 liter IHF buffer - LS
      Stock for 1 liter
    20 mM HEPES pH 7.0 1 M 20 ml
    1 mM EDTA 0.5 M 2 ml
    400 mM NaCl 5 M 80 ml
    10% glycerol 50% 200 ml
    Water   Up to 1 liter

  2. Chill buffer to 4° C
  3. Remove IHF supernatant from -80° C, thaw on ice
  4. Ammonium sulfate to give 50% in 40 ml = 12.52 g
  5. Slowly add ammonium sulfate whilst stirring at 4° C
  6. Once all is in solution, let stir an additional 30 minutes
  7. Transfer to 50 ml centrifuge bottle, spin at 15,000 rpm for 40 minutes
  8. Transfer supernatant to new beaker, add ammonium sulfate to 80% saturation (8.56 g). Add slowly and stir as above.
  9. Spin at 15,000 rpm for 40 minutes
  10. Remove supernatant, redissolve pellet in 1-2 pellet volumes of buffer
  11. Use 2,000 MWCO dialysis tubing, dialyze overnight against LS buffer

Day 8

  1. Thaw samples on ice (about 2 hours)
  2. Set up column (10 mm ID x 10 cm length with flow adapter)
  3. Resuspend heparin, transfer 11 ml to falcon tube
    • Centrifuge at setting 3 for 1 minute (clinical 'fuge)
    • Remove supernatant, add equal amount of IHF buffer - LS and resuspend
    • Repeat wash 4 times
    • Pour resin in pre-wetted column
  4. Set up column with gradient peristaltic pump. Wash 10 volumes (50 ml) at 1 ml/min @ 100% IHF buffer - LS
  5. Load sample at 1 ml/min (about 15 ml)
  6. Wash with 10 bed volumes at 1 ml/min with 100% IHF buffer - LS
  7. Elute at 1 ml/min from 0% to 100% IHF buffer - HS over 50 ml with 1 ml fractions
  8. Quickfreeze fractions in liquid nitrogen

Day 9

  1. Determine IHF fractions by dotting 1 ml of each fraction onto nitrocellulose and doing a western
  2. Run a 10 - 20% protein gel on IHF containing fractions then coomassie stain
  3. Perform nuclease test on fractions
  4. Pool appropriate fractions (22 - 30 in my case = 9 ml), quantitate by OD276
  5. The 9 ml pool ended at 1.2 mg/ml which is fine for us so we didn't concentrate the sample

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