Use either Ni-chelate column from Novagen's
His Bind resin or Clontech's
TALON cobalt column.
The following protocols is for His Bind resin
1) Express and harvest cells. Resuspend
cells in Binding Buffer. Lyse cells.
2) Prepare resin (‾20- 25 ml?) in column - wet column and frit, transfer resin
slurry to column. Wash with water (3 volume), Charge Buffer (5 volumn), Binding
Buffer (3 volume).
3) Load column with cell lysate, at a rate of ‾10 column volumn per hour.
4) Wash column (10 X volumn) with Binding Buffer
5) Wash column (6 X volumn) with Wash Buffer
6) Elute protein (6 X volume) with Elute Buffer (strip buffer can also be used
to elute)
7) To store column, wash with Strip Buffer (3 X volumn) and stored in Strip
Buffer at 4 °C.
1X Buffer
|
reagents |
Binding Buffer |
Wash Buffer |
Elute Buffer |
Strip Buffer |
Charge Buffer |
|
|
Tris-HCl pH7.9 |
20 mM |
20 mM |
20 mM |
20 mM |
|
|
|
Imidazole |
5 mM |
60 mM |
1M |
|
|
|
|
NaCl |
0.5 M |
0.5 M |
0.5 M |
0.5 M |
|
|
|
EDTA |
|
|
|
100 mM |
|
|
|
NiSO4 |
|
|
|
|
50 mM |
|
Purification in Denaturing Condition (protein in inclusion body)
1) Same as before.
2) Centrifuge to collect inclusion body. Decant supernatant, resuspend in
Binding Buffer (sonicate if necessary), centrifuged. Repeat until all trapped
proteins are released.
3) Decant supernatant, resuspend in Binding Buffer + 6 M guanidine or Urea.
Incubate on ice for 1 hour to dissolve.
4) Centrifuged at 39,000 g for 20 min. Filter supernatant (0.45 micron
membrane).
5) Load onto column. Purification same as before, except that all buffer
contain denaturant, and lower imidazole concentration in Wash Buffer (20 mM)
and Elute Buffer (‾300mM) (dilute Wash and Elute Buffer with Binding Buffer).
(Another Method for renaturation of proteins from inclusion body)
Regeneration of Column
- when flow rate is slow and column resin doesn't turn blue-green when charged
1) Wash with 2 vol 6 M guanidine-HCl, 3 vol water.
2) Wash with 1 vol 2% SDS.
3) Wash with 1 vol each of 25%, 50% and 75% ethanol, then 5 vol of 100%
ethanol, followed by 1 vol each of 75%, 50% and 25% ethanol.
4) Wash with 1 vol water, then 5 volume 100 mM EDTA (pH 8.0).
5) Wash with 3 vol water, then 3 vol 20% ethanol.
6) Store at 4 °C.
Note:
1) Do not use ßME, DTT or EDTA in
buffer.
2) Higher amount of imidazole may be used in the wash buffer (100 mM) but some
protein may be eluted.
3) You can elute the protein with salt concentration gradient or the strip
buffer.
4) It's sometimes useful to introduce a couple of glycines between your protein
and the His-tag which allow the his-tag to be fully exposed for effective
binding to resin.
5) See TIBS article "Purification of His-Tag fusion
proteins from Escherichia coli" for more discussion.