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
- 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
- Concentrate the raw protein sample to a volume of < 50 mL
- Filter the protein sample through a 0.22 um vacuum filter
Resin preparation
- Resuspend the resin by inverting the bottle several times (the resin comes as a 50% slurry in 20% EtOH)
- 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
- Spin down resin at 1000 RPM for 3 minutes
- Remove supernatant and discard
- Resuspend resin in 10 resin bed volumes of equilibration/wash buffer
- Spin down resin at 1000 RPM for 3 minutes and remove supernatant
- Repeat steps 5 and 6
Batch binding and washes
- Add the concentrated protein sample to the resin
- Incubate at 4oC for 60 minutes with gentle rocking to keep the resin in suspension
- 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
- Wash - Add 20 resin bed volumes of 1x equilibration/wash buffer to the resin and incubate at 4oC for 10 minutes with gentle rocking
- Spin down resin and remove the supernatant
- Repeat steps 5 and 5
- 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
- Transfer the resuspended resin to a clean 10 mL gravity-flow column with stopper in place
- Allow the resin to settle out of suspension
- Drain the remaining buffer until it reaches the top of the resin bed. Do not let the resin bed dry out
- 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
- 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
- Wash resin with 10 bed volumes of MES buffer, pH 5.0
- Wash resin with 10 bed volumes of ddH20
- Wash resin with 10 bed volumes of 20% EtOH
- Store resin at 4oC in 20% EtOH
Protein analysis
- 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
- 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