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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 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, unless the protein is not binding to the column

'''Protein sample preparation'''<br>
#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