Knight:Purification of His-tagged proteins/Native: Difference between revisions
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*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. | *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. :) | *20 year old spin columns don't work. :) | ||
*The Sauer lab tends to use a higher salt concentration in the lysis, wash and elution buffers. It may give a better wash and may be useful with DNA binding proteins. | |||
==Safety== | ==Safety== |
Revision as of 09:21, 28 September 2006
Overview
Native purifications permit purification of the folded protein potentially at the cost of protein purity.
Materials
- Lysozyme
- Sonicator
Lysis and column equilibration buffer
- 50mM NaH2PO4
- 300mM NaCl
- 10mM imidazole
- can vary between 1mM and 20mM
- pH8.0
Wash buffer
- 50mM NaH2PO4
- 300mM NaCl
- 20mM imidazole
- pH8.0
Elution buffer
- 50mM NaH2PO4
- 300mM NaCl
- 250mM imidazole
- pH8.0
Notes
- Do not use 5X phosphate buffer solution supplied with the kit to make these buffers. It contains Tris.
Procedure
- Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
- The following morning, dilute back the culture 1:60 to the appropriate culture volume (which depends on the expected yield of the protein).
- Maybe try 100mL cultures.
- Harvest the cells by centrifugation at 4000 x g for 15 mins at 4°C.
- Decant supernatant.
- The cell pellet can be stored at -70°C or processed immediately.
- Chill the following on ice ...
- lysis buffer
- wash buffer
- elution buffer
- 2mL eppendorf tube
- Thaw for 15 mins on ice.
- May take longer than 15 mins.
- Transferred to 2mL eppendorf tube.
- Resuspend in 1mL lysis buffer (see above).
- The lysis buffer contains 10mM imidazole to reduce untagged, contaminating proteins. The imidazole concentration can be reduced to either 1-5 mM or increased to 20 mM as appropriate.
- Add 10 μL 100 mg/mL lysozyme to 1 mg/mL final concentration.
- Add a protease inhibitor here?
- Incubate cells on ice for 30 mins.
- Sonicate or homogenize on ice to lyse cells.
- Six times for 10s each time with 5s pauses in between.
- Centrifuge lysate at 10000 x g for 20-30 mins at 4°C to pellet cellular debris. Collect supernatant.
- 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 at 4°C.
- Save 20μL cleared lysate.
- Load 600 μL cleared lysate to Ni-NTA column.
- Centrifuge Ni-NTA column 5 mins at 700 x g with closed lid at 4°C.
- The closed lid increases binding time.
- 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 at 4°C.
- 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 at 4°C.
- 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 at 4°C.
- Most of the protein should elute in this elution step.
- Tranfer to clean 1.5mL eppendorf tube.
- Add 200μL elution buffer.
- Centrifuge Ni-NTA column 2 mins at 700 x g with open lid at 4°C.
- Just in case.
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.
- 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. :)
- The Sauer lab tends to use a higher salt concentration in the lysis, wash and elution buffers. It may give a better wash and may be useful with DNA binding proteins.