CPSC 265
Purification of a 6xHis tagged recombinant lectin using metal affinity chromatography
A. Preparation of metal affinity resin
1. Your TAs will provide you each with 100  l of metal affinity resin slurry in a 1.5 ml micro-centrifuge tube.
2. Centrifuge the resin at high speed for 1 minute to pellet the resin. (Note: the resin pellet should be pink).
3. Using a micropipette, remove and discard the supernatant.
4. Add 200  l of lysis buffer and resuspend the resin pellet by gently by pipetting.
5. Centrifuge the resin at high speed for 1 minute to pellet the resin, remove and discard the supernatant.
B. Metal affinity resin purification
1. Obtain 1 ml of bacterial culture expressing the protein of interest in a 1.5 ml tube.
2. Centrifuge the culture at high speed for 2 min.
3. Keep the cell pellet, you can throw away the supernatant.
4. Place the cell pellet on dry ice for 5 minutes.
5. Remove the tube from the dry ice (freeze-thaw) and then quickly add 500 ul of lysis buffer.
5. Vortex until cell pellet is completely dissolved.
6. Centrifuge at high speed for 5 min to pellet any insoluble debris.
7. Transfer the supernatant into the 1.5 ml tube containing the prewashed metal affinity resin from step five in part A.
8. Mix the sample at room temperature for 10 minutes by slowly rocking the tube by hand (this binds the protein).
9. Centrifuge at high speed for 1 minute to pellet the resin.
10. Carefully remove and discard the supernatant.
11. Add 1 ml of lysis buffer.
12. Invert the tube until the resin is resuspended (this washes off unbound protein).
13. Centrifuge at high speed for 1 min to pellet resin.
14. Remove the supernatant and discard.
15. Repeat wash steps 11-14.
16. Elute the bound 6xHis protein by adding 50  l of 100 mM EDTA (pH 8.0) to the pellet. Vortex briefly.
Note: EDTA strips the bound metal, ensuring elution of all bound 6xHis protein.
17. Add 50  l of 2X SDS loading buffer.
18. Incubate the resin/sample buffer mixtures at 100 °C for 5 minutes in the heating block.
19. Centrifuge briefly at high speed.
20. Load 12  l of each sample onto the SDS-PAGE gel provided by your instructor.
The gel will be run for 30 minutes at 200 volts.
21. The gel is stained as described for the previous lab.

CPSC 265
Solutions Required
Lysis buffer:
50 mM NaH2PO4 (pH 8.0)
10 mM Tris-HCl (pH 8.0)
8M urea
100 mM NaCl
SDS-PAGE sample buffer (2X)
0.125 M Tris-CL pH 6.8
4% SDS
20% glycerol
2% 2-mercaptoethanol
0.01% bromphenol blue
Isopropanol fixing solution
25% (v/v) isopropanol
10% (v/v) acetic acid
65% H
2
O
Rapid Coomassie blue staining solution
10% (v/v) acetic acid
0.001% (w/v) Coomassie brilliant blue G-250 (Bio-Rad)
90% H
2
O
10% Acetic Acid
100mM EDTA, pH 8.0