Swartz:Protocols/Purification of His-tagged proteins: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
mNo edit summary |
mNo edit summary |
||
(One intermediate revision by the same user not shown) | |||
Line 2: | Line 2: | ||
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 | ||
==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== |
Latest revision as of 23:51, 24 February 2013
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