Knight:Purification of His-tagged proteins/Native: Difference between revisions
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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 | ||
* | *Sonicator | ||
===Lysis and column equilibration buffer=== | ===Lysis and column equilibration buffer=== | ||
* | *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 | #*''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°C.'' | #*''The Qiagen protocol didn't specify a temperature so I did 4°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 | #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 | #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. | |||
#Centrifuge lysate at 10000 x ''g'' for 30 mins at | |||
#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. | ||
#Save 20 μL 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 | #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 14:23, 24 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
- 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
- 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.
- The Qiagen protocol didn't specify a temperature so I did 4°C.
- Decant supernatant.
- The cell pellet can be stored at -70°C or processed immediately.
- I stored the pellet at -80°C.
- 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 lysozyme to 1 mg/mL concentration.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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. :)