Protein Purification (Syringe/Pump)

Prepare the column:
Column should always be left in excess buffer. Do NOT let beads dry out!! Column will not dry out by gravity flow alone but because of surface tension of the buffer. But need to be careful when using pump to force liquid over column to never let the liquid run out and pump the column dry.

Uncap column, hook up to either pump or syringe as desired (will take a long time via gravity flow alone). Let column mostly clear of whatever buffer it was in.

Equilibrate column: wash column with at least one column volume of your properly pH’ed buffer to equilibrate.

Run your sample:
Once column is equilibrated, fill column with your sample. Place free end of tube in the rest of your sample (if any) and place beaker w/your sample under the column to form a continuous loop. Set pump to ~ 15. Be sure to watch for a few minutes to make sure it is stable. If you pump too fast, the pressure may blow the top lid off the column and pump your sample all over the floor in your absence.

Allow to cycle for 20-25 minutes to get all of your sample (or as much as will bind) onto the column. CHECK EVERY FIVE MINUTES TO ENSURE COLUMN DOES NOT RUN DRY! Tubes should be coiled 1x or more in the beaker to avoid them falling out and pulling air into the beads.

Once sample is loaded, remove free end of tube from beaker and let drain til mostly through.

Wash column:
Fill column with Wash Buffer. Allow to flow for a few seconds. Place free end of tubing in Wash Buffer beaker, allow pump to flow Wash Buffer over column until all Wash Buffer has been used (save ~1 mL for spectroscopic analysis blank later). Stop column by capping bottom and top once nearly all Wash Buffer has entered column (but do not wait til beads run dry!)

Elute sample:
Fill column with Elution buffer. At this point, incubate the elution buffer for 10 min. Then allow to elute via gravity or use syringe if you are impatient.

For information on regenerating the column: Streptactin Column Regeneration