Knight:Purification of His-tagged proteins/Denaturing

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Line 27: Line 27:
*The lysis buffer requires many drops of 5M NaOH (tens) to bring to pH 8.0.
*The lysis buffer requires many drops of 5M NaOH (tens) to bring to pH 8.0.
*The elution buffer only needs a little (few drops) of 1M HCl to bring to pH 4.5.
*The elution buffer only needs a little (few drops) of 1M HCl to bring to pH 4.5.
 +
*Kathleen suggested supplementing the lysis buffer with 10mM imidazole to prevent nonspecific protein binding to the column.
==Procedure==
==Procedure==

Revision as of 12:55, 29 September 2006

Contents

Overview

Denaturing purifications can often lead to better purity and yield.

Materials

Lysis and column equilibration buffer (Qiagen buffer B)

  • 8 M urea
  • 100 mM NaH2PO4
  • 10 mM Tris Cl
  • 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)
  • When initially made up the solution had a pH of 5.88.
  • The lysis buffer requires many drops of 5M NaOH (tens) to bring to pH 8.0.
  • The elution buffer only needs a little (few drops) of 1M HCl to bring to pH 4.5.
  • Kathleen suggested supplementing the lysis buffer with 10mM imidazole to prevent nonspecific protein binding to the column.

Procedure

  1. Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
  2. The following morning, dilute back the culture 1:60 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. From looking at the culture, it was near but not at saturation but I didn't have time to take OD readings.
  3. 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.
  4. 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.
  5. Decant supernatant.
  6. The cell pellet can be stored at -70°C or processed immediately.
    • I stored the pellet at -80°C.
  7. Thaw for 30 mins.
    • Thaw at room temperature since the next step happens at room temperature.
    • The Qiagen protocol calls for 15 mins, but it was still frozen after 15 mins so I let it thaw for 30 mins.
  8. Transferred to 2mL eppendorf tube.
  9. Resuspend in 1mL Lysis Buffer (see above).
  10. 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.
  11. Centrifuge lysate at 10000 x g for 30 mins at room temperature.
  12. Add 600 μL lysis buffer to Ni-NTA column to equilibrate.
  13. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid to remove equilibration buffer.
  14. Save 20 μL cleared lysate.
  15. Load 600 μL cleared lysate to Ni-NTA column.
  16. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • Repeat this step to load the rest of my cleared lysate?
    • Save flow through.
  17. Add 600 μL wash buffer to Ni-NTA column.
  18. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • Save flow through.
  19. Add 600 μL wash buffer to Ni-NTA column.
  20. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • Save flow through.
  21. Tranfer to clean 1.5mL eppendorf tube.
  22. Add 200μL elution buffer.
  23. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • Most of the protein should elute in this elution step.
  24. Tranfer to clean 1.5mL eppendorf tube.
  25. Add 200μL elution buffer.
  26. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • 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.
  • 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.  :)

Safety

References

  1. Sauer:Purification of His-tagged proteins/Denaturing prep [SauerDenaturingProtocol]
  2. Qiagen Ni NTA Spin Kit manual [QiagenNTAManual]
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