Batch/Gravity-Flow column purification with TALON resin
1. Thoroughly resuspend TALON resin
2. Transfer required amount of resin suspension to a sterile tube that will
accommodate 10 – 20 times the resin bed volume
3. Centrifuge at 700 x g for 2 minutes to pellet resin
4. Remove and discard supernatant
5. Add 10 bed volumes of 1X Equilibration and mix briefly to pre-equilibrate the
resin
6. Recentrifuge at 700 x g for 2 min to pellet the resin. Discard the supernatant
7. Repeat steps 5 and 6.
8. Add clarified lysate to the resin.
9. Rotate at 4oC for 30 minutes
10. Centrifuge at 700 x g for 5 min
11. Remove as much of the supernatant as possible without disturbing the resin pellet.
Save 100 µL and label as “flow through”
12. Add 10 – 20 resin volumes of 1X wash buffer. Gently rotate at 4oC for 15
minutes
13. Centrifuge at 700 x g for 5 minutes
14. Remove supernatant and save 100 µL and label “Wash 1”
15. Repeat steps 12 – 14 and save 100 µL of 2nd wash as “Wash 2”
16. Add 1 resin volume of 1 X Wash buffer to resin and resuspend gently
17. Slowly transfer resin to gravity flow column with end-cap in place and allow resin
to settle out of suspension for 5 minutes
18. Remove end-cap and allow the buffer to drain until it reaches the top of the resin
bed and make sure there are no air bubbles trapped in the resin bed
19. Wash resin with 5 volumes of wash buffer. Save 100µL and label as “Wash 3”
20. Elute the polyhistidine tagged protein by adding 5 resin volumes of Elution
buffer. Collect 500 µL fractions.
21. Analyze fractions by spectrophotometry and by SDS-PAGE
1X Equlibration buffer (pH 7.0)
50 mM sodium phosphate
300 mM NaCl
1X Wash Buffer (pH 7.0)
50 mM sodium phosphate
300 mM NaCl
10 mM imidazole
Imidazole Elution Buffer
50 mM sodium phosphate
300 mM NaCl
150 mM imidazole
pH Elution (pH 5.0)
50 mM sodium acetate
300 mM NaCl
Notes
• Use 2 mL of resin suspension for ~3 mg of anticipated polyhistidine-tagged
protein. 2 mL of homogeneously resuspended resin will provide ~1 mL bed
volume of TALON resin
• Purification of metalloproteins may be affected by elution with imidazole. In this
case, use pH elution.
• If using β-ME in lysis buffer, do not exceed 30 mM. In most cases, only 10 mM
is necessary and excess will reduce the column.
• Resin can be regenerated for multiple purifications. Use either MES or
completely strip and regenerate with EDTA and then CoCl2. (See full protocol)
Figure 1. Talon Resin structure and coordination of cobalt ion.
Figure 2. Typical SDS-PAGE. Make sure to load “flow through”
as well (absent from this gel)
Figure 3. Compatible his-tags for TALON resin
Figure 4. Compatible reagents. EDTA and EGTA will
strip cobalt from column and render it unusable.