Metabolic Labeling of Cells with 35S

1) Transfer to a 24 wells plate the desired colonies.
2) Once the cells are attached (at least 8 hours after tripsinizing them) add ~1 ml of
DME (met-, cys-).
3) Add 3l of Met 35S, Cys 35S trans label.
4) Incubate at least 5 hours at 370C (Put plate(s) inside a tupperware, containing a
beaker with activated charcoal, and close loosely).
5) Collect the media into 1.7 mL tubes and add 5l of the desired antibody.
6) Incubate at least 1 hour using the mixer located at cold room.
7) Centrifuge briefly.
8) Add 100l of a 75:25 protein A:50mM Tris mixture.
9) Incubate at least 1 hour using the mixer located at cold room.
10) Centrifuge briefly.
11) Remove the supernatant using a 5 ml syringe with a 26 1/2 G needle.
12) Add 1 mL of 1 x PBS/0.05% Tween to the beads.
13) Mix by tapping and by inverting it a couple of times.
14) Centrifuge for 30 seconds at 6,000 RPM.
15) Repeat steps 12-14 two more times.
16) Repeat steps 12-14 one more time but instead of adding 1 x PBS/0.05% Tween
add 1 x PBS.
17) Take as much volume as you can out of the beads and add 10l of 50 mM tris and
15l of non-denaturing loading buffer.
18) Boil samples at least 5 minutes.
19) Load as much as you can on an SDS-PAGE (I use 15% Gels) gel.
20) After running the gel incubate it 45 minutes on 30% ETOH:10% HOac.
21) Dry gel for 1.5-2.0 hours at 70 C.
22) Expose O/n to a phosphoimager screen.