Swartz:Protocols/Purification of His-tagged proteins

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Current revision (02:51, 25 February 2013) (view source)
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For purification of Cell-free proteins see https://docs.google.com/document/d/1bSKL1hx6VjR3rMI4UtpCcMEDm7OYm8pnpkQHids4v5g/edit
For purification of Cell-free proteins see https://docs.google.com/document/d/1bSKL1hx6VjR3rMI4UtpCcMEDm7OYm8pnpkQHids4v5g/edit
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=General protocol for His-tag protein purification with 1mL columns=
 
==Buffers==
==Buffers==
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:Buffer C: Same as A with 250mM Imidazole
:Buffer C: Same as A with 250mM Imidazole
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==Cell-free protein synthesis:==
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==General protocol for His-tag protein purification with 1mL columns==
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+
===Protein Production===
 +
====Cell-free protein synthesis:====
:Run cell-free reaction under standard protocols
:Run cell-free reaction under standard protocols
::-For purification, I usually use 1mL CFPS reaction in a 6-well culture plate
::-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
:After completion, add Buffer C so that the sample has a 10mM Imidazole concentration as for Buffer A, the loading buffer
 +
====Cell Prep====
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: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
-
==Purification:==
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===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
:Wash column with 5mL water
:Add 2mL 0.1M NiSO4
:Add 2mL 0.1M NiSO4
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:Load sample, collecting flowthrough
:Load sample, collecting flowthrough
:Wash with 10mL Buffer B, collecting in 5mL fractions
:Wash with 10mL Buffer B, collecting in 5mL fractions
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::(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)
+
::(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
:Elute with 3mL Buffer C, collecting in 1mL fractions
:Final wash with 2mL Buffer B
:Final wash with 2mL Buffer B
Line 37: Line 58:
:Run about 8uL on gel (or less for flowthrough fraction) to check for elutions to pool
:Run about 8uL on gel (or less for flowthrough fraction) to check for elutions to pool
 +
 +
==Notes==

Current revision

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


Contents

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

Notes

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