Difference between revisions of "Swartz:Protocols/Purification of His-tagged proteins"
From OpenWetWare
m |
m |
||
| Line 3: | Line 3: | ||
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== | ==Buffers== | ||
| Line 11: | Line 10: | ||
:Buffer C: Same as A with 250mM Imidazole | :Buffer C: Same as A with 250mM Imidazole | ||
| − | ==Cell-free protein synthesis:== | + | ==General protocol for His-tag protein purification with 1mL columns== |
| − | + | ===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==== | ||
| + | :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 | :Wash column with 5mL water | ||
:Add 2mL 0.1M NiSO4 | :Add 2mL 0.1M NiSO4 | ||
| Line 26: | Line 47: | ||
: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 | ||
| − | + | ::(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== | ||
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
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
