Knight:Purification of His-tagged proteins/Native

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==Overview==
==Overview==
-
Native purifications permit purification of the folded protein.
+
Native purifications permit purification of the folded protein potentially at the cost of protein purity.
==Materials==
==Materials==
*Lysozyme
*Lysozyme
-
*RNase A (optional)
+
*Sonicator
-
*DNase A (optional)
+
-
 
+
===Lysis and column equilibration buffer===
===Lysis and column equilibration buffer===
-
*5mM imidazole
+
*50 MM NaH<sub>2</sub>PO<sub>4</sub>
 +
*300mM NaCl
 +
*10mM imidazole
**can vary between 1mM and 20mM
**can vary between 1mM and 20mM
 +
*pH8.0
===Wash buffer===
===Wash buffer===
-
 
+
*50 MM NaH<sub>2</sub>PO<sub>4</sub>
 +
*300mM NaCl
 +
*20mM imidazole
 +
*pH8.0
===Elution buffer===
===Elution buffer===
-
 
+
*50 MM NaH<sub>2</sub>PO<sub>4</sub>
 +
*300mM NaCl
 +
*250mM imidazole
 +
*pH8.0
===Notes===
===Notes===
-
 
+
*Do not use 5X phosphate buffer solution supplied with the kit to make these buffers.
==Procedure==
==Procedure==
#Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
#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).
#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 200mL 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.
#*''The Qiagen protocol didn't specify a temperature so I did 4&deg;C.''
#*''The Qiagen protocol didn't specify a temperature so I did 4&deg;C.''
Line 33: Line 40:
#*''May take longer than 15 mins.''
#*''May take longer than 15 mins.''
#Transferred to 2mL eppendorf tube.
#Transferred to 2mL eppendorf tube.
-
#Resuspend in 2-5mL Lysis Buffer (see above) per gram wet weight.
+
#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 lysozyme to 1 mg/mL concentration.
#Add lysozyme to 1 mg/mL concentration.
-
#Incubate cells cells on ice for 30 mins.
+
#Incubate cells on ice for 30 mins.
-
#Optional: If the lysate is very viscous, add 10 &mu;g/mL RNase A and 5 &mu;g/mL DNase I and incubate on ice for 10-15 mins.
+
#Sonicate or homogenize on ice to lyse cells.
-
#*Or draw the lysate through a narrow gauge blunt-ended syring needle several times.
+
#*''Six times for 10s each time with 5s pauses in between.''
-
 
+
#Centrifuge lysate at 10000 x ''g'' for 20-30 mins at 4&deg;C to pellet cellular debris.  Collect supernatant.
-
 
+
-
#Centrifuge lysate at 10000 x ''g'' for 30 mins at room temperature.
+
#Add 600 &mu;L lysis buffer to Ni-NTA column to equilibrate.
#Add 600 &mu;L 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.
-
#Save 20 &mu;L cleared lysate.
+
#Save 20&mu;L cleared lysate.  
#Load 600 &mu;L cleared lysate to Ni-NTA column.
#Load 600 &mu;L cleared lysate to Ni-NTA column.
-
#Centrifuge Ni-NTA column 2 mins at 700 x ''g'' with open lid.
+
#Centrifuge Ni-NTA column 5 mins at 700 x ''g'' with closed lid.
 +
#*''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.''

Revision as of 17:23, 24 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

  • 50 MM NaH2PO4
  • 300mM NaCl
  • 10mM imidazole
    • can vary between 1mM and 20mM
  • pH8.0

Wash buffer

  • 50 MM NaH2PO4
  • 300mM NaCl
  • 20mM imidazole
  • pH8.0

Elution buffer

  • 50 MM NaH2PO4
  • 300mM NaCl
  • 250mM imidazole
  • pH8.0

Notes

  • Do not use 5X phosphate buffer solution supplied with the kit to make these buffers.

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.
    • The Qiagen protocol didn't specify a temperature so I did 4°C.
  4. Decant supernatant.
  5. The cell pellet can be stored at -70°C or processed immediately.
    • I stored the pellet at -80°C.
  6. Thaw for 15 mins on ice.
    • May take longer than 15 mins.
  7. Transferred to 2mL eppendorf tube.
  8. 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.
  9. Add lysozyme to 1 mg/mL concentration.
  10. Incubate cells on ice for 30 mins.
  11. Sonicate or homogenize on ice to lyse cells.
    • Six times for 10s each time with 5s pauses in between.
  12. Centrifuge lysate at 10000 x g for 20-30 mins at 4°C to pellet cellular debris. Collect supernatant.
  13. Add 600 μL lysis buffer to Ni-NTA column to equilibrate.
  14. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid to remove equilibration buffer.
  15. Save 20μL cleared lysate.
  16. Load 600 μL cleared lysate to Ni-NTA column.
  17. Centrifuge Ni-NTA column 5 mins at 700 x g with closed lid.
    • The closed lid increases binding time.
    • Repeat this step to load the rest of my cleared lysate?
    • Save flow through.
  18. Add 600 μL wash buffer to Ni-NTA column.
  19. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • 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.
    • Save flow through.
  22. Tranfer to clean 1.5mL eppendorf tube.
  23. Add 200μL elution buffer.
  24. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • Most of the protein should elute in this elution step.
  25. Tranfer to clean 1.5mL eppendorf tube.
  26. Add 200μL elution buffer.
  27. 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.
  • 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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