Anti-FLAG
Co-immunoprecipitation
Transient transfection:
Day 1 (preferably early):
Trypsinize a confluent 10-cm plate of cells and plate 0.5 ml
cells plus 1.5 ml medum per 3.5-cm plate (for multiple plates, mix cells after
trypsinizing and plate from the mixture to get more homogenous plates).
Day 2:
When plates are 50-80% confluent transfect the cells using
Lipofectamine as described by manufacturer.
For each 3.5-cm plates use:
8 µl Lipofectamine; mix with 100 µl O-MEM
1-2 µg plasmid; mix with 100 µl O-MEM
Mix Lipofectamine and plasmid mixtures.
Leave at RT 15-45 min.
Add 800 µl O-MEM.
Remove medium from cells
Wash carefully with 1 ml O-MEM.
Add Lipofectamine/plasmid mixture (1 ml total) to cells.
Incubate 5-24 hours in incubator
Remove transfection mixture.
Add DMEM/10% FBS carefully.
Incubate 24-48 h.
Immunoprecipitation:
Day 4:
Wash cells carefully with 2 ml PBS.
Add 1 ml PBS and scrape off cells using a rubber policeman.
Transfer to eppendorf tube.
Spin 1,000 rpm, 10 min (not faster!).
Remove PBS.
Resuspend in ice-cold 400 µl hypotonic gentle lysis buffer
and transfer to eppendorf tube.
Incubate on ice, 10 min.
Add NaCl to 150 mM. Incubate on ice 5 min.
Spin 14,000 rpm, 4C, 15 min.
Transfer 50 µl supernatant to 50 µl of SDS LB (Total extract
control). Store at -20C.
Transfer remainder of supernatant to tube containing 40 µl
anti-FLAG-M2 agarose slurry, which has already been washed twice with 1 ml
NET-2 (1,000 rpm, 1 min for each wash).
Nutate at 4C, ≥4h.
Wash beads 8 times with 1 ml NET-2 (ice-cold). Spin 1,000
rpm, 1 min for each wash. (Try to remove as much wash buffer as possible
without removing beads in each wash).
After last wash, remove all wash buffer with pipette. Add 20
µl SDS LB to beads (pellets).
Western blot:
Run SDS gel.
Cut four pieces of 3MM papers and a piece of nitrocellulose
membrane at 8x11 cm (or 15x20cm for large gel).
Prepare 1 liter (or 4 liters for large gel) of transfer
buffer: 20% methanol,3.1 g/l Trizma base, 14.4 g/l Glycine. Pre-cool in cold
room.
Transfer gel to a piece of 3MM and place on top of a piece
of 3MM under transfer buffer. Place membrane on top of gel followed by two
pieces of 3MM. Remove bubbles by rolling a pippette over the
"sanwich".
Transfer to transfer box and fill with transfer buffer.
Run at 80V, 3-4 hours in cold room.
(Optional: Check membrane with Ponceau Red. Wash with Blot
buffer)
Transfer membrane to Blot buffer/10% milk.
4C, o/n.
Incubate at RT 1-4 h with primary antibody in Blot buffer/5%
milk.
Wash 2x5 min with Blot buffer.
Incubate at RT 1-4 h with secondary antibody in Blot
buffer/5%milk.
Wash 3x10 min with blot buffer.
Drip off, but don't dry out.
In dark room:
Incubate with chemiluminescence reagent 1 min.
Drip off, but don't dry out!!
Wrap in Saram Wrap. Use autorad tape (or other means to
orient film)
Expose to film 1 min.
Develop, and evaluate for longer or shorter exposure.
(Signal has a hal-life of 5-10 mins.)
Stripping:
Incubate in water 15 min.
Incubate in 0.1M NaOH, 15 min.
Incubate in water 15 min.
Block in Blot buffer/10% milk, 4C, o/n.
Buffers:
Hypotonic gentle lysis buffer:
10 mM Tris-HCl pH 7.5
10 mM NaCl
10 mM EDTA
0.5% Triton-X100
1 mM PMSF
1 µM aprotinin
1 µM leupeptin
0.1 units/µl RNasin
NET-2:
50 mM Tris-HCl pH7.5
150 mM NaCl
0.05% Triton-X100
SDS LB:
see "Sambrook et al."
Blot buffer:
100 mM Tris HCl pH 7.5
150 mM NaCl
0.1% Tween 20