3 hr method:
1. Two days after transfection, rinse cells carefully but quickly with Hanks to remove the FCS. (With 293Ts this step is omitted if they look as though they may slip off the plate)
2. Add 2mls met/cys-free DMEM + 1% dialyzed FCS + P/S/G. Leave for 1 hr.
3. Replace with 1.0 ml fresh Met/Cys-free DMEM/1% dialyzed FCS + P/S/G containing 125 mCi/ml 35S-Met for 3hrs.
4. Remove labeling medium and save for reuse.
1. As late a possible the night before the IP (usually 24 hr after beginning the transfection), remove medium and replace with 1 ml Met/Cys-free DMEM / 1% dialyzed FCS + P/S/G containing 125mCi/ml 35S-Met. Begin the IP as early as possible the next a.m.
0.5% TRITONX-100 LYSIS AND IP
1. Remove labeling medium as above and wash 1X with PBS/PMSF.
2. Add 1 ml of Extraction buffer (0.5% TX-100, 25mMTris pH7.4, 5 mM EDTA, 1mM PMSF, 10 ug/ml Aprotinin) to each plate and incubate on ice for 30 to 45 min. with mild shaking.
3. Scrape cells in extraction buffer and transfer to eppendorfs. Spin for 15 min at high speed in the cold room to pellet all material not soluble in TX-100.
4. Transfer sup't to new eppendorfs and proceed as in step 7 below.
RIPA SOLUBLE/INSOLUBLE FRACTIONS: (This is used to determine at what point proteins oligomerise or become attached to the cytoskeleton.)
1. Add 300l-1ml of RIPA buffer / 35mm dish to cells on dish - leave on rocker in cold for 10mins.
2. Scrape cells into eppendorf and spin 50,000g for 10 mins.
3. Collect supt =soluble fraction.
4. Process pellet as described above = insoluble fraction.
SDS-LYSIS and IP
Prepare: Ice buckets and ice trays Scrapers Chilled & labeled eppendorfs Tubes and boxes for radioactive waste + Screen + Napkins Wash Sepharose-prot A beads 1X in PBS. Resuspend at 1:1 in PBS. Lysis and Soln A + inhibitors -store 4C and on ice :
Lysis buffer Soln A 25mM Tris pH 7.4 50mM Tris pH 7.4 3mM EDTA 1mM EDTA 150mM NaCl 150mM NaCl 1% SDS 2% TritonX-100
Wash Buffer OR RIPA Wash Buffer:
1% Triton X100 20mM Tris 7.5
1% deoxycholate 0.15M NaCl 0.1% SDS 1mM EDTA
1M NaCl 1% NP-40
15mM Tris pH 7.5
5mM EDTA (some additinally add 0.1mM DTT; 25mM KCl; 2mM EGTA)
Immediately before use add 1/1000 Aprotinin (10ug/ml) 1/100 PMSF (1mM)
Stocks are stored at -20C Aprt = 10mgs/ml in H2O; PMSF = 100mM in ethanol
1. Remove and discard Labeling medium, taking care not to dislodge the cells.
2. Scrape cells with a rubber policeman into an eppendorf in 1 ml PBS/1mM PMSF . Microfuge 5 min at 1000 rpm in refrigerated microcentrifuge to gently collect cells.
3. Discard supt in Radiactive Waste and add 200l of lysis buffer
4. Sonicate 2X for 15 sec stop 6.
5. Boil 2 mins. Spin 30 sec. Dilute with 1ml of Soln A to bring the final SDS conc to below 0.2% SDS and complexe it into micelles. Otherwise the SDS concentration is too high for the antibody to work.
6. Spin 15 mins at 4oCin eppendorf to pellet any insoluble junk. Pellet, if any, should be very, very small compared to the cell pellet.
7. To eppendorfs add: 30l beads using cut tips antibody (1-3l flag; 8-10l myc; 5l RAM) 1 ml supt
8. Rotate 3 hr to O/N in the cold room.
9. Spin down beads and wash 6X in wash buffer
10. Boil beads in 30 to 50l Laemmli and load on gel