Knight:Purification of MBP-tagged proteins: Difference between revisions
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(New page: ==Overview== A protein purification procedure based on proteins fused to maltose binding protein (MBP). ==Materials== *[http://www.neb.com/nebecomm/products/productE8000.asp pMAL Protein...) |
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<font color=red>in progress!</font> | |||
==Overview== | ==Overview== | ||
A protein purification procedure based on proteins fused to maltose binding protein (MBP). MBP tagged expression systems have the advantage that MBP has been shown to enhance the solubility of expressed proteins facilitating purification. It assumed that you are already have a construct expressing your MBP fusion. | |||
A protein purification procedure based on proteins fused to maltose binding protein (MBP). | |||
==Materials== | ==Materials== | ||
*[http://www.neb.com/nebecomm/products/productE8000.asp pMAL Protein Fusion and Purification System] | *[http://www.neb.com/nebecomm/products/productE8000.asp pMAL Protein Fusion and Purification System] | ||
*Column buffer | |||
**20 mM Tris-HCl (HEPES is also ok) | |||
**200 mM NaCl | |||
**1 mM EDTA | |||
**0.5mM TCEP is likely ok | |||
**10 % glycerol is ok but not required | |||
**Zinc sulfate is likely also ok | |||
==Procedure== | ==Procedure== | ||
# | #Grow a 5mL culture overnight in LB + glucose + antibiotic | ||
#Dilute back 100-fold into 80mL culture of LB + glucose + antibiotic | |||
#Grow at 37°C to an OD600nm of 0.6. | |||
#Divide the cell cultures into two aliquots of 40 mL each. | |||
#Harvest the cells by centrifugation at 4000 x ''g'' for 10 minutes. | |||
#Discard supernatant. | |||
#Resuspend pellet in 5mL of column buffer. | |||
#Freeze the cells in a dry ice-ethanol bath. | |||
#Thaw in icy water. | |||
#Repeat freeze thaw cycles 2-3 more times to lyse cells. | |||
#*''NEB recommends sonication for < 15 seconds while cells are in a ice water bath. Usually takes 2 minutes.'' | |||
#*''Optional: monitor the release of the protein with a Bradford assay by adding 10 μL sonicate to 1.5mL of Bradford reagent and mixing.'' | |||
#*''Save 13μL cell lysate for SDS-PAGE gel.'' | |||
#Centrifuge at 9000 x ''g'' at 4°C for 20 minutes. | |||
#Decant the supernatant and save on ice. | |||
#*''Save 13μL crude extract for SDS-PAGE gel.'' | |||
#Resuspend pellet in 5 mL column buffer (insoluble fraction). | |||
#*''Save 13μL insoluble matter fraction for SDS-PAGE gel.'' | |||
#Place 200 μL of amylose resin in microfuge tube. | |||
#Spin briefly in microcentrifuge. | |||
#Resuspend the resin in 1.5mL column buffer. | |||
#Microcentrifuge briefly. | |||
#Discard supernatant. | |||
#Resuspend the resin in 200 μL column buffer. | |||
#Mix 50 μL crude extract with 50 μL amylose resin slurry. | |||
#Incubate 15 mins on ice. | |||
#Microcentrifuge for 1 minute. | |||
#Remove supernatant and discard. | |||
#Wash pellet with 1mL column buffer. | |||
#Microcentrifuge for 1 minute. | |||
#Remove supernatant and discard. | |||
#*''Save 13μL of amylose bound to protein for SDS-PAGE gel.'' | |||
==Notes== | ==Notes== | ||
#[http://www.neb.com/nebecomm/products/faqproductE8000.asp NEB FAQ on pMAL kit] | #[http://www.neb.com/nebecomm/products/faqproductE8000.asp NEB FAQ on pMAL kit] | ||
Revision as of 15:00, 2 March 2007
in progress!
Overview
A protein purification procedure based on proteins fused to maltose binding protein (MBP). MBP tagged expression systems have the advantage that MBP has been shown to enhance the solubility of expressed proteins facilitating purification. It assumed that you are already have a construct expressing your MBP fusion.
Materials
- pMAL Protein Fusion and Purification System
- Column buffer
- 20 mM Tris-HCl (HEPES is also ok)
- 200 mM NaCl
- 1 mM EDTA
- 0.5mM TCEP is likely ok
- 10 % glycerol is ok but not required
- Zinc sulfate is likely also ok
Procedure
- Grow a 5mL culture overnight in LB + glucose + antibiotic
- Dilute back 100-fold into 80mL culture of LB + glucose + antibiotic
- Grow at 37°C to an OD600nm of 0.6.
- Divide the cell cultures into two aliquots of 40 mL each.
- Harvest the cells by centrifugation at 4000 x g for 10 minutes.
- Discard supernatant.
- Resuspend pellet in 5mL of column buffer.
- Freeze the cells in a dry ice-ethanol bath.
- Thaw in icy water.
- Repeat freeze thaw cycles 2-3 more times to lyse cells.
- NEB recommends sonication for < 15 seconds while cells are in a ice water bath. Usually takes 2 minutes.
- Optional: monitor the release of the protein with a Bradford assay by adding 10 μL sonicate to 1.5mL of Bradford reagent and mixing.
- Save 13μL cell lysate for SDS-PAGE gel.
- Centrifuge at 9000 x g at 4°C for 20 minutes.
- Decant the supernatant and save on ice.
- Save 13μL crude extract for SDS-PAGE gel.
- Resuspend pellet in 5 mL column buffer (insoluble fraction).
- Save 13μL insoluble matter fraction for SDS-PAGE gel.
- Place 200 μL of amylose resin in microfuge tube.
- Spin briefly in microcentrifuge.
- Resuspend the resin in 1.5mL column buffer.
- Microcentrifuge briefly.
- Discard supernatant.
- Resuspend the resin in 200 μL column buffer.
- Mix 50 μL crude extract with 50 μL amylose resin slurry.
- Incubate 15 mins on ice.
- Microcentrifuge for 1 minute.
- Remove supernatant and discard.
- Wash pellet with 1mL column buffer.
- Microcentrifuge for 1 minute.
- Remove supernatant and discard.
- Save 13μL of amylose bound to protein for SDS-PAGE gel.
Notes
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Contact
- Reshma Shetty wrote up this protocol.
