Swartz:Protocols/Purification of His-tagged proteins

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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=
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==Buffers==
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:Buffer A: 10mM Imidazole, 50mM NaPhosphate pH 8, 300mM NaCl
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:Buffer A.5: Same as B with 30mM Imidazole
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:Buffer B: Same as A with 50mM Imidazole
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:Buffer C: Same as A with 250mM Imidazole
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==Cell-free protein synthesis:==
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:Run cell-free reaction under standard protocols
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::-For purification, I usually use 1mL CFPS reaction in a 6-well culture plate
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:After completion, add Buffer C so that the sample has a 10mM Imidazole concentration as for Buffer A, the loading buffer
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==Purification:==
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:Wash column with 5mL water
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:Add 2mL 0.1M NiSO4
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:Wash with 5mL water
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:Equilibrate with 5mL Buffer A
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:Load sample, collecting flowthrough
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: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)
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:Elute with 3mL Buffer C, collecting in 1mL fractions
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:Final wash with 2mL Buffer B
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:Wash column with 5mL water
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:Regenerate with 5mL 0.5M EDTA
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:Equilibrate with 5mL Buffer A (only because pH of my EDTA solution is very high)
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:Wash with 5mL water
 +
:Store in 3mL 20% EtOH
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:Run about 8uL on gel (or less for flowthrough fraction) to check for elutions to pool

Revision as of 02:16, 15 May 2012

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

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
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