Purification of 6xHis Tagged -GalactosidaseProtocol

Affinity Column Purification Protocol using B-Per 6xHis Fusion Protein Purification Kit©, product #78100 by Pierce, Inc. (

  1. Defrost the frozen pellet of E.coli BL21(pET-14b) cells prepared in the previous lab session and add 10.0 mL of B-Per Protein Extraction Reagent™, a mild anionic detergent (the exact ingredients of which are proprietary), to the centrifuge bottle. Add 20L of DNAase (to achieve a concentration of 1 unit/mL from the 5000units/ml stock) to the B-Per and cells. Resuspend the cells by vortex mixing and pipetting up and down until the pellet is off the side of the bottle and all clumps are gone. Try to avoid bubbles. This should take about 5 minutes total, of alternately mixing and letting the mixture sit in your ice bucket. Your goal is to dissolve the cell walls and membranes which will lyse the cells and allow degradation of the DNA. Ask your instructor to check your suspension before proceeding to step 2. If you have obvious clumps, the cells are less likely to be lysed sufficiently.
  2. Shake the suspension gently for 10 minutes at room temperature using a platform shaker. During this time the lysed cells will release their contents and soluble bacterial proteins will be dissolved in the lysate. Be sure to break up any obvious clumps with gentle vortexing.
  3. Carefully pour the entire suspension into a Corex tube fitted with a rubber adapter and centrifuge for 15 minutes at 10,500 rpm in the Sorval refrigerated centrifuge at 4°C using an SS-34 rotor. You will need to make a balance tube with water. Be sure and record in your lab notebook the speed as g force (use the chart near the centrifuges to make the conversion).
  4. Pour the supernatant only into a 15 mL graduated conical tube. This solution of dissolved proteins in B-Per reagent is your cell-free or Crude Extract (CE). The pellet is bacterial cell debris that can be dumped into your waste container. The Corex tubes are NOT disposable. Please rinse them out and leave them on your bench or return them to your instructor.
  5. Determine and record the total volume of the crude extract (CE) using the volume markings on the tube. Pipet 600 µL of CE into a microcentrifuge tube labeled with your team color, lab day, initials and "CE" or “Crude Extract.” Store the aliquot on ice. At the end of lab today, you will give what remains of this aliquot to your instructor to freeze for use next time.
  6. Add 1000µL (1mL) of the Nickel-Chelated Agarose to the volume of CE remaining in the 15mL conical tube. It is essential that the agarose suspension be properly mixed before removing an aliquot, so mix it well by swirling or gentle vortexing just before removing it from the stock.
  7. Shake the Agarose-CE mixture gently for 10 minutes at room temperature on the platform shaker. Make sure the cap is secured tightly. Centrifuge for 5 minutes at top speed in a clinical, benchtop centrifuge.
  8. Using a Pasteur pipette, carefully remove and discard the supernatant solution into the provided waste container. Do not try to pour off the supernatant! The 6xHis-tagged proteins should now be bound to the nickel-chelated agarose beads at the bottom of the tube.
  9. Add 3.0 mL of Wash Buffer #1®to the agarose beads in the conical tube, and resuspend using a pipette. Do not vortex. Gently shake the suspension at room temperature for 5 minutes on the platform shaker. Centrifuge for 3 minutes in a clinical, benchtop centrifuge. Carefully remove supernatant and discard.
  10. Add 3.0 mL of Wash Buffer #2® to the centrifuge tube and resuspend the agarose using a pipette. Do not vortex. Gently shake the suspension at room temperature for 5 minutes on the platform shaker. Centrifuge for 3 minutes in a clinical, benchtop centrifuge. Carefully remove supernatant and discard.
  11. Add 250µL of Wash Buffer #2® to the agarose, resuspend completely and transfer all of this suspension from the centrifuge tube to a B-PERTM Spin Column that has been placed into one of the special collection tubes provided. To insure that the agarose beads don’t get stuck in the pipet tip of your P1000, cut about a 1/4 inch off the end of it with a single edged razor blade. Centrifuge for 2 minutes at maximum speed in a microcentrifuge. Discard the collection tube with the flow-through.
  12. Place the spin column in a new collection tube and add 500µL of Elution Buffer containing imidazole. Mix the agarose and the buffer in the spin column using the NOT DISPOSABLE plastic mixer provided by your instructor. Incubate the spin column at room temperature for 5 minutes. During this time, most of the 6x His-tagged protein will be eluted from the nickel-chelated agarose into the buffer. After the 5 minute incubation period, recover the protein by centrifuging for 2 minutes at top speed in a microcentrifuge. Your purified fraction will be the flow-through in the collection tube. If you have considerably less than 500µL, respin the column to collect more volume.
  13. Take the spin column out of the collection tube and discard the spin column. Do not discard the material in the collection tube. Using a Pasteur pipette, transfer all of the contents containing the purified β-galactosidase to a new microfuge tube with volume markings. Record the volume in your notebook! Label the tube to indicate that this is the purified -galactosidase fraction (e.g., "PF β-gal"), and save it in your ice bucket.

DO NOT DISCARD ANY OF THE CRUDE EXTRACT OR PURIFIED FRACTION!

You are now ready to assay both the Crude Extract and Purified Fraction for total protein, using a modified Bradford Dye Assay (Bio-Rad assay). You will later assay both CE and PE for β-galactosidase activity (enzyme function) using a different assay.