This protocol describes the expression of the migration-related protein focal adhesion kinase (FAK) in Sf9 insect cells.
Method Article
Production of FAK in baculovirus system
https://doi.org/10.1038/nprot.2009.17
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
You are reading this latest protocol version
focal adhesion kinase expression
This protocol describes the expression of the migration-related protein focal adhesion kinase (FAK) in Sf9 insect cells.
Binding Buffer 1
1XPBS pH7.5
200mM NaCl
2mM β-ME
Wash Buffer
1XPBS pH7.5
200mM NaCl
2mM β-ME
10% glycerol
20mM Imidazole
Elution Buffer
1XPBS pH7.5
200mM NaCl
2mM β-ME
10% glycerol
100mM Imidazole
Binding Buffer 2
1XPBS pH7.5
200mM NaCl
2mM β-ME
10% glycerol
Gel Filtration Buffer
1XPBS pH7.5
250mM NaCl
2mM β-ME
Expression:
2x500ml Sf9 cells at 2x106 cell/ml
10ml virus
27 °C
130rpm
Grow until cells are 30% dead
Harvest: 2.5krpm, 15min, 4 °C. Resuspend in 20ml PBS/200mM NaCl
LN2 freeze
Purification:
Thaw 2x500ml cell pellets. Add PI tablets w/o EDTA (crush 2 Roche tablets in 2ml PBS/200mM NaCl. Add 1ml/500ml cell pellet).
Sonicate cells 15-20sec 5-6 times on ice. Check cells under microscope to see if cells have busted open.
Spin 19krpm, 60min, 4 °C.
Equilibrate 2ml column with Binding buffer 1.
Load column with supernatant.
Wash column with 50ml binding buffer 1.
Wash column with 50ml binding buffer 2.
Wash column with 30ml wash buffer.
Elute with elution buffer. Collect fractions. Pool peak. If protein starts to crash out, use 5 M NaCl to bring back into solution up to 500 mM total.
Concentrate protein to go onto S75 gel filtration column.
Gel filtration with 1xPBS/250mM NaCl/2mM β-ME. (with the S75 column you can load 500 μl sample at a time roughly at 3-5mg/ml)
Critical: if you need to concentrate the protein you will need to go up in NaCl and possible glycerol to stabilize the protein.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
You are reading this latest protocol version