LOCAL

Lab Protocols

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

  1. Equilibrate column in Wash Buffer

  2. Load sample

  3. Wash until UV reaches baseline

  4. Shallow gradient of Elution Buffer.  NOTE: One can usually bump off different forms of his-tag protein (depending on his-tag exposure).

  5. 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