Swartz:Protocols/Purification of His-tagged proteins

See Purification_of_His-tagged_proteins and https://docs.google.com/document/d/1OqY94AYARJD84spfuFVZ_YX1Ypa-i717sxUUJU2GJ9Y/edit

For purification of Cell-free proteins see https://docs.google.com/document/d/1bSKL1hx6VjR3rMI4UtpCcMEDm7OYm8pnpkQHids4v5g/edit

General protocol for His-tag protein purification with 1mL columns

Buffers

Buffer A: 10mM Imidazole, 50mM NaPhosphate pH 8, 300mM NaCl

Buffer A.5: Same as B with 30mM Imidazole

Buffer B: Same as A with 50mM Imidazole

Buffer C: Same as A with 250mM Imidazole

Cell-free protein synthesis:

Run cell-free reaction under standard protocols

-For purification, I usually use 1mL CFPS reaction in a 6-well culture plate

After completion, add Buffer C so that the sample has a 10mM Imidazole concentration as for Buffer A, the loading buffer

Purification:

Wash column with 5mL water

Add 2mL 0.1M NiSO4

Wash with 5mL water

Equilibrate with 5mL Buffer A

Load sample, collecting flowthrough

Wash with 10mL Buffer B, collecting in 5mL fractions

::(Alternatively, washes can be done with 5mL Buffer A, 5mL A.5, and 5mL B to test binding or with 5mL Buffer A if protein is known to elute early)

Elute with 3mL Buffer C, collecting in 1mL fractions

Final wash with 2mL Buffer B

Wash column with 5mL water

Regenerate with 5mL 0.5M EDTA

Equilibrate with 5mL Buffer A (only because pH of my EDTA solution is very high)

Wash with 5mL water

Store in 3mL 20% EtOH

Run about 8uL on gel (or less for flowthrough fraction) to check for elutions to pool