A. Laganowsky
Eisenberg Laboratory
7-6-2008
Adapted From: Protein Production and Purification (2008) Nature Methods. 5:135-146
Native His-Tag Purification
Lysis Buffer (1 Liter)
50mM NaH2PO4 M.W. 137.99 6.9 g
0.5 M NaCl M.W. 58.44 29.22 g
10 mM Imidazole M.W. 68.08 0.68 g
5% Glycerol 50 mL
Adjust pH to 8.0
Before use add to final conc.
10U/mL Benzonase
Protease Inhibitor cocktail
37.5 uL / 100mL BME (14M Stock) final 5mM
Wash Buffer (1 Liter)
50mM NaH2PO4 M.W. 137.99 6.9 g
0.3 M NaCl M.W. 58.44 17.54 g
20 mM Imidazole M.W. 68.08 1.36 g
5% Glycerol 50 mL
Adjust pH to 8.0
Before use add to final conc.
37.5 uL / 100mL BME (14M Stock) final 5mM
Elution Buffer (1 Liter)
50mM NaH2PO4 M.W. 137.99 6.9 g
0.3 M NaCl M.W. 58.44 17.54 g
500 mM Imidazole M.W. 68.08 34.0 g
5% Glycerol 50 mL
Adjust pH to 8.0
Before use add to final conc.
37.5 uL / 100mL BME (14M Stock) final 5mM
Brief protocol
Equilibrate column in Wash Buffer
Load sample
Wash until UV reaches baseline
Shallow gradient of Elution Buffer. NOTE: One can usually bump off different forms of his-tag protein (depending on his-tag exposure).
Wow!
NOTE: I usually add between 0.1-0.5M Guanidine HCL final concentration to clarified cell lysate. This works well for protein chaperones.
Affinity Enhancing Buffer (1 Liter / 500 mL)
100mM NaH2PO4 M.W. 137.99 13.8 g / 6.9 g
10mM Tris-base M.W. 121.14 1.2 g / 0.6 g
6M Guanidine Hydrochloride M.W. 95.5 573 g / 286.5 g
Adjust pH to 8.0