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

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

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

Protein Production

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

Cell Prep

Add single colony or glycerol stock to 5ml LB with 25ug/ml chloramphenicol in addition to any resistance markers on plasmids

Shake at 37C for 12-14 hours overnight

Autoclave 1L LB flasks

Add entire contents of overnight cultures to 1L flasks (in addition to antibiotics) and shake at 37C

Grow to an OD of ~0.5 and induce with 1ml of 1M IPTG

less IPTG can be used

growth usually takes around 4 hours

induction is usually for 4 hours at 37C but time and temperature can be varied depending on protein sensitivity

Spin down cell pellets, measure mass, and store at -80C

Extract prep:

Thaw pellets in 15mL Buffer A

Prepare cell homogenizer by washing with water, NaOH, and EtOH as per instructions on machine

Resuspend pellets using hand homogenizer

Add 80uL of 250mM PMSF to resuspended pellets

Lyse by flowing through homogenizer 1 pass at >20,000psi

Add ~2,200U of DNaseI

Spin down in 30ml tubes for 30min at 13,000rpm

Discard pellet and allow supernatant to thin on bench

usually takes at least an hour

add more Buffer A if still viscous

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