Knight:Purification of His-tagged proteins/Denaturing

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*Solid urea to make up an 8M solution takes up a lot of volume so be conservative on how much H <sub>2</sub>O you start with (maybe 50% of final volume)
*Solid urea to make up an 8M solution takes up a lot of volume so be conservative on how much H <sub>2</sub>O 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.  Perhaps supplement the other buffers as well?
*Kathleen suggested supplementing the lysis buffer with 10mM imidazole to prevent nonspecific protein binding to the column.  Perhaps supplement the other buffers as well?
 +
*The urea should be freshly prepared and deionized prior to use.
 +
*The buffers should each be degassed before use.
==Procedure==
==Procedure==

Revision as of 21:46, 13 December 2006

Contents

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
  • 1 mM TCEP (add just before use?)

Wash buffer (Qiagen buffer C)

  • 8 M urea
  • 100 mM NaH2PO4
  • 10 mM Tris Cl
  • 10 mM imidazole (recommended by Kathleen, 9/27/2006, also use here?)
  • pH 6.3
  • 10 mM TCEP (add just before use?)

Elution buffer (Qiagen buffer E)

  • 8 M urea
  • 100 mM NaH2PO4
  • 10 mM Tris Cl
  • 10 mM imidazole (recommended by Kathleen, 9/27/2006, also use here?)
  • pH 4.5
  • 1 mM TCEP (add just before use?)

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. Perhaps supplement the other buffers as well?
  • The urea should be freshly prepared and deionized prior to use.
  • The buffers should each be degassed before use.

Procedure

  1. Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
  2. 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.
  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.
    • 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.
  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 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.
  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.

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. Some people also seem to use 0.5-1mM TCEP with these columns. TCEP appears to more compatible with NTA columns for his-tagged protein purification.
  • Even when doing denaturing purifications, add 10mM imidazole to solutions to help with washing out non His tagged proteins.

Troubleshooting and optimization

  • Deionize urea solutions prior to use.
  • Degas all buffers.

Safety

References

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