In vivo metabolic labeling of BM macrophage with 35S-Met/Cys and
immunoprecipitation
1. Wash cells with Met-Cys- (MMCS) MEM
2. Incubate in MMCS MEM for 15 min at 37oC
3. Label for 3 hours with the labeling medium in plexiglasss box with charcoal filter
paper
4. Wash cells with PBS (cold) 2x
5. Extract protein with solubilization buffer (0.5 ml per 6 cm plate ) – on ice for 30
min
6. Scrape cells, spin at 14 K at 4oC for 15 min, transfer to a fresh tube
7. Save some for TCA ppt., protein determination or total lysate lane on gel
Labelling buffer
1. MEM Met-Cys- (+ L-Gln)
2. 10% dialyzed FBS
3. 35S-Met/ Cys labeling mix (250 µCi/ ml)
RIPA buffer
150 mM NaCl
1% NP-40
0.5% deoxycholate
0.1% SDS
50 mM Tris.HCl pH 7.5
protease inhibitor cocktail
Variables:
1. Treat with LPS ( 0.1 µg/ml) for 1 hr or not
2. wash cells and follow the labeling regime (above)
3. label for 3 hrs ( + LPS)
4. lyse and IP
IP protocol:
1. Add 20 µl of protein-A-sepharose beads + 1 µl NRS into cleared lysate and rotate
for 1 hr at 4 degrees C
2. Spin 5 k 15 sec, and remove supe to a new tube
3. Add 1 µl antibody (casp-11 or COX-2)
4. Add 20 µl protein-A-sepharose beads and and rotate for 1 hr at 4 degrees C
5. Spin 5 k 30 sec, wash 6x with the binding buffer
6. Be careful in the washes not to disturb the pellet
7. After the final wash take all the liquid using a fine bore pipet tip
8. Elute in ~ 50 µl of Laemelli sample buffer