A common protocol for the purification of polyhistidine tagged proteins uses an Immobilized metal affinity column (IMAC). This protocol for affinity purification is a generalization that utilizes Amersham sepharose beads and nickel ions.
Buffers Binding Buffer - pH 8.0 with monosodium phosphate and NaOH, and 20mM Imidazole to reduce nonspecific binding.
Wash Buffer - pH 8.0 with monosodium phosphate and NaOH, and 60mM Imidazole to reduce nonspecific binding.
Elution Buffer - pH 8.0 with monosodium phosphate and NaOH, and 400mM Imidazole to reduce nonspecific binding. You can optionally use a pH 6.0 buffer that elutes the metal ions from the agarose beads, but this introduces metal ion contamination to the elution.
Column Preparation 1. In 4 C cold room, set up the column on a vertical support.
Flush the column with cold ddH2O. Leave some water in the column.
Resuspend the Amersham sepharose. Transfer the sepharose to the column using a pipette with the tip cut off.
Open the outlet for the column to allow the sepharose to pack at the bottom. Do not let the sepharose dry out.
Wash the column with 5 volumes of ddH2O. Drain the last wash to just above the sepharose.
Add two column volumes of 0.1M NiSO4. Drain the NiSO4.
Wash the column with 5 column volumes of ddH2O until the sepharose is blue-green.
Wash the column with 5 column volumes of binding buffer until the sepharose is blue.
Thaw cell pellet from 250 ml culture.
Resuspend pellet in 15 ml Binding buffer and 1.0 mM PMSF (150 ul of 100mM).
a. Binding buffer is pH 8.0 with monosodium phosphate and NaOH, and 20mM Imidazole to reduce nonspecific binding.
Add the resuspension to prechilled oakridge tubes.
Sonicate the cell suspension 10 seconds at a time, with 10 seconds in between sonications. Repeat 7 times.
Centrifuge the lysate in SS34 rotor at 18,000 rpm (39,000 RPM) for 20 minutes at 4 oC.
Filter the lysate supernatant through a 0.22 um syringe filter.
Immobilized Metal Ion Affinity Chromatography
Apply lysate to the prepared column. Collect the flow through in a tube and reapply to the column.
Repeat step 1 two more times.
a. Optionally, you can save 100 ul of the flow through for analyzing initial binding conditions. If so, add 2X laemmli buffer to the 100 ul and freeze at -20 oC.
Prepare 50 ml (10 column volumes) of wash buffer with 0.1 mM PMSF (500 ul).
Add the wash buffer to the column, draining 5 ml fractions.
a. Optionally save these fractions by freezing them at -80 oC.
For fractions 2, 5, and 8, transfer 10 ul of the fraction to a microfuge tube and add 2X Laemmli buffer. Freeze these tubes at - 20 oC.
Prepare 10 ml elution buffer (10 column volumes) with 0.1mM PMSF (250 ul).
Add 5 ml elution buffer to the column and allow it to incubate for 5 minutes before allowing it to wash through.
Collect the elution in 1 ml fractions using 13x100 mm disposable glass tubes. Save all fractions by freezing them at -80 oC.
a. Optionally save 10 ul of each elution fraction by adding 10 ul 2X Laemmli buffer in a microfuge tube. Freeze these tubes at -20 oC.
Wash the column with 10 volumes of binding buffer. If storing the column for a long period of time, wash the column with ddH2O and store the sepharose in 20% ethanol.