O3-73 cell lysates were prepared in RIPA buffer (2×107 cells in 300 μL) and sonicated for 40 cycles (30s, vortexed each 10 cycles, Diagenode Bioruptor Pico). For IP-westerns, each lysate was incubated with rabbit anti-Gal4DBD antibody (1 μg, Santa Cruz sc-577×) attached to Dynabeads M-280 sheep anti-rabbit IgG (50 μL) as described by the bead manufacturer. For conventional western blotting, sonicated lysates were used directly (10 μL). Whole cell or IP lysates were fractionated on 12% PAGE gels, transferred to PVDF membranes, incubated with detection antibodies and visualized by ECL substrate (Thermo Fisher Scientific #34087). Primary antibodies were used at a concentration of 1 μg/mL for protein detection and obtained from the following sources: mouse anti-Gal4DBD antibody (clone RK5C1; Santa Cruz sc-510), rabbit anti-ETS1 (clone N276; Santa Cruz sc-111), mouse anti-GAPDH (clone G-9; Santa Cruz sc-365062), rabbit anti-RUNX1 C terminus (Dr. Takeshi Egawa) (42 (link)), mouse anti-RUNX1 NRDB domain (clone A-2; Santa Cruze sc-365644). Secondary antibodies, diluted 1:104, were goat anti-rabbit IgG-HRP (Santa Cruz sc-2004) and goat anti-mouse IgG-HRP (Promega W4021).
Protocol detail
Immunoprecipitation and Western Blotting
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Corresponding Organization : Washington University in St. Louis
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Variable analysis
independent variables
- The concentration of anti-Gal4DBD antibody used for immunoprecipitation (1 μg)
- The volume of Dynabeads M-280 sheep anti-rabbit IgG used for immunoprecipitation (50 μL)
- The levels of Gal4DBD, ETS1, GAPDH, RUNX1 C terminus, and RUNX1 NRDB domain proteins detected by western blotting
- The number of O3-73 cells used for cell lysate preparation (2×10^7 cells)
- The volume of RIPA buffer used for cell lysis (300 μL)
- The number of sonication cycles (40 cycles)
- The duration of sonication (30s) and vortexing (10 cycles)
- The type of PAGE gel used for protein fractionation (12%)
- The type of membrane used for protein transfer (PVDF)
- The type of ECL substrate used for protein detection
- Rabbit anti-Gal4DBD antibody (Santa Cruz sc-577×) used for immunoprecipitation
- No controls were explicitly mentioned in the provided information
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