Knight:Purification of His-tagged proteins/Native

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(Procedure)
Line 32: Line 32:
#The following morning, dilute back the culture 1:60 to the appropriate culture volume (which depends on the expected yield of the protein).
#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.''
#*''Maybe try 100mL cultures.''
-
#Harvest the cells by centrifugation at 4000 x ''g'' for 15 mins.
+
#Harvest the cells by centrifugation at 4000 x ''g'' for 15 mins at 4°C.
-
#*''The Qiagen protocol didn't specify a temperature so I did 4°C.''
+
#Decant supernatant.
#Decant supernatant.
#The cell pellet can be stored at -70°C or processed immediately.
#The cell pellet can be stored at -70°C or processed immediately.
-
#*''I stored the pellet at -80°C.''
+
#Chill the following on ice ...
 +
#*lysis buffer
 +
#*wash buffer
 +
#*elution buffer
 +
#*2mL eppendorf tube
#Thaw for 15 mins on ice.
#Thaw for 15 mins on ice.
#*''May take longer than 15 mins.''
#*''May take longer than 15 mins.''
Line 42: Line 45:
#Resuspend in 1mL lysis buffer (see above).
#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.''
#*''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 μ/mL 100 mg/mL lysozyme to 1 mg/mL final concentration.
+
#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.
#Incubate cells on ice for 30 mins.
#Sonicate or homogenize on ice to lyse cells.
#Sonicate or homogenize on ice to lyse cells.
Line 48: Line 52:
#Centrifuge lysate at 10000 x ''g'' for 20-30 mins at 4°C to pellet cellular debris.  Collect supernatant.
#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.
#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.
+
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid to remove equilibration buffer at 4°C.
#Save 20μL cleared lysate.  
#Save 20μL cleared lysate.  
#Load 600 μL cleared lysate to Ni-NTA column.
#Load 600 μL cleared lysate to Ni-NTA column.
-
#Centrifuge Ni-NTA column 5 mins at 700 x ''g'' with closed lid.
+
#Centrifuge Ni-NTA column 5 mins at 700 x ''g'' with closed lid at 4°C.
#*''The closed lid increases binding time.''
#*''The closed lid increases binding time.''
#*''Repeat this step to load the rest of my cleared lysate?''
#*''Repeat this step to load the rest of my cleared lysate?''
#*''Save flow through.''
#*''Save flow through.''
#Add 600 μL wash buffer to Ni-NTA column.
#Add 600 μL wash buffer to Ni-NTA column.
-
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.
+
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid at 4°C.
#*''Save flow through.''
#*''Save flow through.''
#Add 600 μL wash buffer to Ni-NTA column.
#Add 600 μL wash buffer to Ni-NTA column.
-
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.
+
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid at 4°C.
#*''Save flow through.''
#*''Save flow through.''
#Tranfer to clean 1.5mL eppendorf tube.
#Tranfer to clean 1.5mL eppendorf tube.
#Add 200μL elution buffer.
#Add 200μL elution buffer.
-
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.
+
#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.''
#*''Most of the protein should elute in this elution step.''
#Tranfer to clean 1.5mL eppendorf tube.
#Tranfer to clean 1.5mL eppendorf tube.
#Add 200μL elution buffer.
#Add 200μL elution buffer.
-
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.
+
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid at 4°C.
#*''Just in case.''
#*''Just in case.''

Revision as of 20:43, 25 September 2006

Contents

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

  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).
    • Maybe try 100mL cultures.
  3. Harvest the cells by centrifugation at 4000 x g for 15 mins at 4°C.
  4. Decant supernatant.
  5. The cell pellet can be stored at -70°C or processed immediately.
  6. Chill the following on ice ...
    • lysis buffer
    • wash buffer
    • elution buffer
    • 2mL eppendorf tube
  7. Thaw for 15 mins on ice.
    • May take longer than 15 mins.
  8. Transferred to 2mL eppendorf tube.
  9. 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.
  10. Add 10 μL 100 mg/mL lysozyme to 1 mg/mL final concentration.
  11. Add a protease inhibitor here?
  12. Incubate cells on ice for 30 mins.
  13. Sonicate or homogenize on ice to lyse cells.
    • Six times for 10s each time with 5s pauses in between.
  14. Centrifuge lysate at 10000 x g for 20-30 mins at 4°C to pellet cellular debris. Collect supernatant.
  15. Add 600 μL lysis buffer to Ni-NTA column to equilibrate.
  16. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid to remove equilibration buffer at 4°C.
  17. Save 20μL cleared lysate.
  18. Load 600 μL cleared lysate to Ni-NTA column.
  19. 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.
  20. Add 600 μL wash buffer to Ni-NTA column.
  21. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid at 4°C.
    • Save flow through.
  22. Add 600 μL wash buffer to Ni-NTA column.
  23. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid at 4°C.
    • Save flow through.
  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 at 4°C.
    • Most of the protein should elute in this elution step.
  27. Tranfer to clean 1.5mL eppendorf tube.
  28. Add 200μL elution buffer.
  29. 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.  :)

Safety

References

  1. Qiagen Ni NTA Spin Kit manual [QiagenNTAManual]
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