HisPur Cobalt Resin Protein Purification Protocol

This is a combined protocol from Clontech's protein purification products manual and the HisTALON gravity column purification kit manual. The lysis portion follows the protein purification manual and the purification by gravity flow is from the kit. The native protein purification protocol using the TALON resin is normally a batch/gravity flow.

Grow 500ml bacterial culture and induce at OD = 0.5 with 1mM IPTG

Spin down 500ml culture at 5,000g for 10 mins. Discard supernatant and weigh dry pellet. Proceed to protein purification or store at -80deg.

Before you begin:

• Prechill all solutions on ice 30 mins before you begin protocol

Protein Purification

1) Thaw pellet on ice for 10 mins

2) Add 40ml ICE COLD Equalibration buffer and resuspend pellet by vortexing <SAVE SOME FOR LYSIS EFFICIENCY CHECK>

3) Add 30mg (0.75mg/ml) lysozyme powder directly to the bacterial resuspension.

4) Add 4ul Benzonase (400U/ul -> 1ul/10ml) + 400ul of 100X protease inhibitor cocktail + 400ul 100mM PMSF (in isopropanol)

5) Incubate on oribital shaker with gentle shaking on ICE for 30 mins

6)Turn on Floor centrifuge to prechill to 4deg

7) Sonicate on ICE at: 50% duty cycle, power=10 (10sec pulse/30sec rest) X3

*under a light microscope, compare bacteria before and after lysis+sonication. 90% of cells should be fragmented. If not, repeat sonication.

8) Spin in JA-17 at 13,000rpm (15,000g) for 30 mins at 4deg

9) Harvest supernatant. Freeze pellet at -20deg. <SAVE SOME PRERUN LYSATE>

10) Add 2ml of HisPur slurry to a clean column. Add the slurry gently to the side of the column via a transfer pipet to ensure that there are no air bubbles. If there are air bubbles trapped, try to dislodge them.

11) Equalibrate the column with 10ml ICE COLD equilibration buffer. Allow to flow through.

12) Load the cleared lysate through the column

13) Wash  column with 8ml of   ICE COLD equilibration buffer

14) Wash  column with 8ml of   ICE COLD wash buffer <SAVE SOME BEADS>

15) Elute with (5) 1ml elutions

Regenerate of Column

1. add 4ml 6M guanidinium+ 1% tritonX-100 ph5.0
2. add 5 ml h2o
3. add 5 ml 20mM MES+0.1M NaCl ph5.0
4. add 5ml h20
5. store resin in 20% ethanol+0.1% Na Azide at 4deg  

The HisPur needs to be completely regenerated with CoCl2 before use

100X protease inhibitor cocktail

1X = 800nM Aprotinin, 20uM Leupeptin

Buffers

To make wash buffer: 660ul elution buffer + 9.34 equalibriation buffer