Production of FAK in baculovirus system
This protocol describes the expression of the migration-related protein focal adhesion kinase (FAK) in Sf9 insect cells.
Binding Buffer 1
Binding Buffer 2
Gel Filtration Buffer
2x500ml Sf9 cells at 2x106 cell/ml
1) Grow until cells are 30% dead
2) Harvest: 2.5krpm, 15min, 4 °C. Resuspend in 20ml PBS/200mM NaCl
1) Thaw 2x500ml cell pellets. Add PI tablets w/o EDTA (crush 2 Roche tablets in 2ml PBS/200mM NaCl. Add 1ml/500ml cell pellet).
2) Sonicate cells 15-20sec 5-6 times on ice. Check cells under microscope to see if cells have busted open.
3) Spin 19krpm, 60min, 4 °C.
4) Equilibrate 2ml column with Binding buffer 1.
5) Load column with supernatant.
6) Wash column with 50ml binding buffer 1.
7) Wash column with 50ml binding buffer 2.
8) Wash column with 30ml wash buffer.
9) 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.
10) Concentrate protein to go onto S75 gel filtration column.
11) 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.
12) SDS PAGE on purification. Do protein concentration. LN2 freeze protein.
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Posted 05 Feb, 2009
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