Protocol describes expression and purification of GIT1 in SF9 cells.
Method Article
GIT1 (full length) protein purification (Baculovirus)
https://doi.org/10.1038/nprot.2009.19
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GIT1
Protocol describes expression and purification of GIT1 in SF9 cells.
1X Binding Buffer (High Salt)
5mM Imidazole
500mM NaCl
10% Glycerol
3mM DTT
20mM Tris-HCl, pH 7.9
Elution Buffer (Medium Salt)
500mM Imidazole
250mM NaCl
10% Glycerol
3mM DTT
20mM Tris-HCl, pH 7.9
1X Binding Buffer (Medium Salt)
5mM Imidazole
250mM NaCl
10% Glycerol
3mM DTT
20mM Tris –HCl, pH 7.9
Dialysis Buffer (Low Salt)
20mM Tris pH7.9
250mM NaCl
10% Glycerol
5mM DTT
1X Wash Buffer (Medium Salt)
20mM Imidazole
250mM NaCl
10% Glycerol
3mM DTT
20mM Tris-HCl pH 7.9
Purification:
Quick thaw 1 L cell pellet. Add 2 mM PMSF, 2 Roche PI tablets w/o EDTA. Sonicate until cells have lysed (3x20sec, on ice 20sec in between).
Add MgCl2 to 10mM. Add 20µl Benzenase. Rock for 60min 4°C until the mixture is really runny. Save 10µl for running on gel.
Spin: 16K, 15 min, 30 ml tubes. Save supernatant and pellet. Save 10µl of each for running on a gel.
Meantime, wash Ni-NTA beads with binding buffer. Do a 1-2ml column.
Load supernatant on column. Save flow through. Save 10µl for gel.
Wash 50ml binding buffer (BB) high salt, then 50ml BB medium salt.
Wash 50ml wash buffer (20mM Imidazole).
Elute with 500mM Imidazole buffer.
Dialyze.
Run SDS PAGE. Measure protein concentration.
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