Knight:Purification of His-tagged proteins/Denaturing: Difference between revisions
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*20 year old spin columns don't work. :) | *20 year old spin columns don't work. :) | ||
*These columns can tolerate up to 20mM β-mercaptoethanol or 1mM dithiothreitol. | *These columns can tolerate up to 20mM β-mercaptoethanol or 1mM dithiothreitol. | ||
*Even when doing denaturing purifications, add 10mM imidazole to solutions to help with washing out non His tagged proteins. | |||
==Safety== | ==Safety== |
Revision as of 14:54, 27 November 2006
Overview
Denaturing purifications can often lead to better purity and yield.
Materials
Lysis and column equilibration buffer
- 8 M urea
- 100 mM NaH2PO4
- 10 mM Tris Cl
- 10 mM imidazole (recommended by Kathleen, 9/27/2006)
- pH 8.0
Wash buffer (Qiagen buffer C)
- 8 M urea
- 100 mM NaH2PO4
- 10 mM Tris Cl
- pH 6.3
Elution buffer (Qiagen buffer E)
- 8 M urea
- 100 mM NaH2PO4
- 10 mM Tris Cl
- pH 4.5
Notes
- Solid urea to make up an 8M solution takes up a lot of volume so be conservative on how much H 2O you start with (maybe 50% of final volume)
- Kathleen suggested supplementing the lysis buffer with 10mM imidazole to prevent nonspecific protein binding to the column.
Procedure
- Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
- The following morning, dilute back the culture 1:50 to the appropriate culture volume (which depends on the expected yield of the protein).
- Since I don't know what yield to expect, I arbitrarily did 50mL cultures assuming that my protein yield would be low and let it grow most of the day. I try to catch the cultures around OD600nm 0.6.
- Verify pH of lysis, wash and elution buffers. Adjust if necessary.
- Dissociation of urea can lead to changes in pH. The pH definitely needs to be checked prior to using the solutions.
- Harvest the cells by centrifugation at 4000 x g for 15 mins.
- The Qiagen protocol didn't specify a temperature so I did 4°C.
- Decant supernatant.
- The cell pellet can be stored at -70°C or processed immediately.
- I stored the pellet at -80°C.
- Thaw for 30 mins.
- Can thaw at room temperature since the next step happens at room temperature. However, proteases will be less active if the pellet is thawed on ice.
- The Qiagen protocol calls for 15 mins, but it was still frozen after 15 mins so I let it thaw for 30 mins.
- Transferred to 2mL eppendorf tube.
- Resuspend in 1mL Lysis Buffer (see above).
- Incubate cells with agitation for 1 hr at room temperature.
- Use an orbis shaker on the bench to do this temp (usually kept in 37° incubator). Note that the shaker moves during shaking.
- Centrifuge lysate at 10000 x g for 30 mins at room temperature.
- Add 600 μL lysis buffer to Ni-NTA column to equilibrate.
- Centrifuge Ni-NTA column 2 mins at 700 x g with open lid to remove equilibration buffer.
- Save 20 μL cleared lysate.
- Load 600 μL cleared lysate to Ni-NTA column.
- Centrifuge Ni-NTA column 5 mins at 700 x g with closed lid.
- I typically repeat this step to load the rest of my cleared lysate.
- Save flow through.
- Add 600 μL wash buffer to Ni-NTA column.
- Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
- Save flow through.
- Add 600 μL wash buffer to Ni-NTA column.
- Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
- Save flow through.
- Tranfer to clean 1.5mL eppendorf tube.
- Add 200μL elution buffer.
- Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
- Most of the protein should elute in this elution step.
Notes
- Sauer lab uses a Qiagen Ni-NTA resin but this protocol uses spin columns. (Smaller scale purification).
- Using the Qiagen Ni-NTA resin may be preferable for proteins with low yields.
- A lower pH may be needed for elution. For instance, aggregates should elute at pH 4.5 whereas monomers generally elute at pH 5.9.
- Contaminating proteins tend to be less of an issue in bacteria because there are few proteins with neighboring histidines that tend to bind to the column.
- 20 year old spin columns don't work. :)
- These columns can tolerate up to 20mM β-mercaptoethanol or 1mM dithiothreitol.
- Even when doing denaturing purifications, add 10mM imidazole to solutions to help with washing out non His tagged proteins.