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

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