Purification of 6xHis Tagged -Galactosidase Protocol
Affinity Column Purification Protocol using B-Per 6xHis Fusion Protein
Purification Kit©, product #78100 by Pierce, Inc. (http://wwwpiercenet.com):
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
4C 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 on the wall near
the centrifuges to make the conversion).
4. Pour the supernatant only into a 15 ml graduated conical centrifuge tube. This
solution of dissolved proteins in B-Per reagent is your cell free or Crude Extract. 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 the CE into a microfuge tube labeled with your
team color, lab day, initials and "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 Crude Extract
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 centrifuge.
8. Using a Pasteur pipette, carefully remove and discard the supernatant solution into
a waste container provided. 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 at
2500rpm. 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. 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 0.5ml, 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 pipet, transfer all of
the contents of this collection tube containing the purified beta-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 ("PF -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 also assay both for betagalactosidase activity using a different assay that measures ONP production.