Wittrup:His-Tag Proteins/Nickel Column Purification (suppliers, troubleshooting, etc.)

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His-Tag protein purification

Adapted from BD Talon Batch/Column purification protocol

Materials

  • BD Talon metal affinity resin (available from Clontech)
  • Equilibration / Wash buffer (50 mM sodium phosphate, 300 mM NaCl, pH 7.0 - 8.0)
  • Colum wash buffer (50 mM sodium phosphate, 300 mM NaCl, 5 mM imidazole, pH 7.0)
  • Elution buffer (50 mM sodium phosphate, 300 mM NaCl, 150 mM imidazole, pH 7.0)
  • 10 mL gravity flow column with stopper

Note: Increasing the pH of the equilibration/wash buffer to 8.0 will increase binding of your target protein to the resin, but may also increase non-specific binding of other proteins. We typically use pH 7.0 for all buffers

Protein sample preparation

  1. Re-equilibrate the raw secretion supernatant in 1x equilibration/wash buffer by dialysis or concentration/dilution. Check the pH of the sample to ensure that it is between 7.0 and 8.0
  2. Concentrate the raw protein sample to a volume of < 50 mL
  3. Filter the protein sample through a 0.22 um vacuum filter

Resin preparation

  1. Resuspend the resin by inverting the bottle several times (the resin comes as a 50% slurry in 20% EtOH)
  2. Pipette 2-4 mL of resin slurry (1-2 mL of resin) into a 50 mL tube. The resin has a capacity of ~ 3 mg of his-tagged protein per mL resin. If you have more protein in your sample, split the resin between 2 or more tubes
  3. Spin down resin at 1000 RPM for 3 minutes
  4. Remove supernatant and discard
  5. Resuspend resin in 10 resin bed volumes of equilibration/wash buffer
  6. Spin down resin at 1000 RPM for 3 minutes and remove supernatant
  7. Repeat steps 5 and 6

Batch binding and washes

  1. Add the concentrated protein sample to the resin
  2. Incubate at 4oC for 60 minutes with gentle rocking to keep the resin in suspension
  3. Spin down resin and pipette off protein supernatant without disturing the resin. Save the protein supernatant in case the protein didn't bind the resin
  4. Wash - Add 20 resin bed volumes of 1x equilibration/wash buffer to the resin and incubate at 4oC for 10 minutes with gentle rocking
  5. Spin down resin and remove the supernatant
  6. Repeat steps 5 and 5
  7. Resuspend the resin in one bed volume of equilibration/wash buffer

Column wash and elution
These steps are typically performed at room temperature but should be done at 4oC if you have an unstable protein

  1. Transfer the resuspended resin to a clean 10 mL gravity-flow column with stopper in place
  2. Allow the resin to settle out of suspension
  3. Drain the remaining buffer until it reaches the top of the resin bed. Do not let the resin bed dry out
  4. Wash the column once with 5 bed volumes of column wash buffer. The column wash buffer has a low concentration of imidazole which helps wash off non-specifically bound proteins
  5. Elute the protein with 5 bed volumes of elution buffer. Collect the eluate in 1 mL fraction

Resin regeneration
The resin can be washed and reused if desired

  1. Wash resin with 10 bed volumes of MES buffer, pH 5.0
  2. Wash resin with 10 bed volumes of ddH20
  3. Wash resin with 10 bed volumes of 20% EtOH
  4. Store resin at 4oC in 20% EtOH

Protein analysis

  1. Measure the UV absorbance at 280 nm of the elution fractions to determine the concentration of purified protein. The majority of purified protein typically elutes in the second 1 mL fraction. The imidazole in the elution buffer will produce a small abs280 reading which must be accounted for when determining protein concentrations
  2. Analyze the elution fractions by SDS-PAGE to check the purity of eluted protein. Also run the raw supernatant and wash fractions to see if any protein is being lost at other steps
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