Purifying 6xHis-Tagged Proteins from E. Coli by Immobilized Metal Affinity Chromatograpy (IMAC) under Native Conditions

SUPPLIES:

Equipment:

  • Nutator or Rocking platform
  • Test Tube rack or equivalent method for holding columns (stand clamp, tape, etc.)
  • Microcentrifuge or Floor Centrifuge capable of 14k RCF + tubes as needed

Consumables:

Buffers and Solutions:

  • Appropriate Buffer solutions:
    • Phosphate buffers are usually the safest choice; Tris and carbonate can cause issues with the Ni++ resin.
    • Buffers should either be made with sterile stock solutions or filter sterilized after preparation
    • Use sterile mqH2O for all solutions

  • Lysis / Binding Buffer:
    • 50mM Sodium Phosphate dibasic (Na2HPO4)
    • 300mM NaCl
    • 10mM Imidazole
    • pH -> 8.0 w/ HCl
    • **1x EDTA-Free Protease Inhibitors (e.g. HALT, Thermo-Pierce Cat. No. 78439)
    • **>250U/mL Benzonase nuclease (Sigma Cat. No. E1014) or equivalent
    • **1mg/mL Lysozyme

**=Add these components just before use

  • Wash Buffer 1:
    • 50mM Sodium Phosphate dibasic (Na2HPO4), pH 8.0
    • 300mM NaCl
    • 20mM Imidazole
    • pH -> 8.0 w/ HCl

  • Wash Buffer 2:
    • 50mM Sodium Phosphate dibasic (Na2HPO4), pH 8.0
    • 500mM NaCl
    • 20mM Imidazole
    • pH -> 8.0 w/ HCl

  • Elution Buffer:
    • 50mM Sodium Phosphate dibasic (Na2HPO4), pH 8.0
    • 300mM NaCl
    • 250mM Imidazole
    • pH -> 8.0 w/ HCl

STEP 1: LYSIS

  1. ) Thaw cell pellets on ice; prepare Lysis Buffer by adding Protease Inhibitors to 1x, Benzonase, and lysozyme (usually easiest to just add powdered lysozyme directly to buffer).
  2. ) Add lysis buffer to cell pellet and resuspend by gently swirling or pipetting. Want to avoid foaming so don’t vortex vigorously. Good rule of thumb is 2mL of lysis buffer to 50mL of expression culture (for E. Coli in LB or 2XYT) or 5mL per gram of wet pellet weight.
  3. ) Allow cells to lyse for 30min @ 37degC.
    • Some people do this step on ice, so might be a good idea if you are worried about stability of your protein. Of course, your protein just spent several hours growing at 37C
  4. ) Vortex Cells for ~2sec to help break up cell walls
    • Lysate shouldn’t be snotty at this point if you added nuclease; if you have trouble with this it may help to add more nuclease or pass the lysate through a 20g needle 10-15 times
  5. ) Transfer lysate to an appropriate centrifuge tube; if 2-5mL of lysate you can use 2mL micro centrifuge tubes. If you have larger volumes use SS-34 tubes or equivalent.
  6. ) Centrifuge lysate @ 14,000xg for 30min to pellet cell debris. Can be done at RT or 4degC depending on the centrifuge; in my experience doesn’t make much difference.
  7. ) Carefully decant supernatant (Cleared Lysate) into a fresh tube.
    • save a 15uL sample of cleared lysate for gel later

STEP 2: IMAC PURIFICATION W/ GRAVITY COLUMN

  1. ) [OPTIONAL] Preparing the column: If your columns come with fritted disc inserts, add a small amount of buffer to the (capped!) column and push the first disc all the way to the bottom with a pasteur pipet, like so: [PICTURE] Then leave the cap in place and pour off any excess buffer. Should be a small amount of buffer (and no bubbles) in the void space below the disc)
  2. ) Transfer the cleared lysate from STEP 1 to the column (WITH BOTTOM CAP ATTACHED!!!)
  3. ) Gently mix the Ni-NTA agarose resin to make a slurry and add directly to the lysate.
    • Good rule of thumb is 500mL slurry (250mL packed volume) per 2mL of lysate for well-expressed proteins, but you may need to adjust this for unusually abundant (if you see lots of protein in the flow-through) or down for poorly expressed proteins (if your purification looks dirty, i.e. has lots of contaminating proteins when viewed on a gel)
    • Slurry settles quickly, so mix GENTLY in between each samples for most consistent results
    • If the small amount of EtOH from the resin storage buffer is a concern:
      • Insert the bottom fritted disc into the gravity column, gently add slurry to the column
      • Allow storage buffer to drain, then rinse w/5 column volumes (CV) of Wash Buffer #1.
      • Cap the bottom of the column, add lysate, cap & parafilm the top of the column, and continue w/ step 4
  4. ) Allow lysate to bind the resin for 30min at RT with gentle agitation (e.g. Nutator, slow rocking, slow rotating)
  5. ) Place the column upright over a 50-mL conical to collect the flow through. A ring stand and clamp works well for a single column; if doing multiple columns at once they can be lined up in a wire test-tube rack and propped up with pipet tip boxes like so: [PICTURE]
  6. ) Remove cap from TOP of column; if using fritted discs to hold the resin, GENTLY push the top disc through the liquid in the column with a pasteur pipet, trapping the resin in between. Try to avoid trapping air between the disc and the resin, and GENTLY push the disc down to the level of the resin bed. Too much pressure will result in columns running slowly.
  7. ) Remove the BOTTOM cap and allow the liquid to pass out of the column. Collect this fraction as the flow-through (F/T) and add 15uL to 5uL of SDS Page sample buffer for a gel later.
  8. ) Add 5-10 resin bed volumes of Wash Buffer #1 to column(s) and allow to pass through. Collect this fraction as Wash #1 and save 15uL to run a gel later.
  9. ) Add 10-20 resin bed volumes of Wash Buffer #2 to column(s) and allow to pass through. Collect this fraction as Wash #2 and save 15uL to run a gel later.
  10. ) Set up 5-10 1.7mL eppendorf tubes to collect elusions fractions
  11. ) Add 500uL of Elution Buffer to the column and collect the eluate in a 1.7mL tube. Repeat 5-10 times for a total of 2.5-5mL of eluate.
  12. ) Test a small sample of each fraction by adding to 100uL of Bradford reagent (or running on an SDS-PAGE gel), and pool the fractions with the highest concentration of protein.
  13. ) Run an SDS-PAGE Gel with at least the Lyaste, Flow Through, Washes 1 & 2, and Pooled Eluate for each sample. His-tag-purified protein samples will still have small amounts of other contaminating proteins visible on a gel, but should be much more homogeneous than the lysate.

[GEL PICTURE]

-- Main.ColinBrown - 25 Aug 2016

 Barrick Lab  >  ProtocolList  >  Protocols6xHisTagProteinPurification

Topic revision: r1 - 25 Aug 2016 - 20:06:22 - Main.ColinBrown
 
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