Runx2 primary mouse antibody

LICOR Odyssey Imaging system allows for quantitative determination of intracellular protein expression levels of cells growing in 96 well culture plates. These In Cell Western blotting (ICW) experiments were performed in cells growing in HTS plates transfected with siRNA library constituents. For ICW experiments after the desired culture period, cells growing in HTS plates were removed from the incubator and cells were washed with warm PBS and fixed with 4% paraformaldehyde solution for 15 minutes followed by washing and permeabilizing with 0.1% TritonX100 in PBS 5 times each. Then the plate was blocked using Odyssey blocking buffer for 1 hour and incubated with Runx2 primary mouse antibody (Santa Cruz Biotechnology, Dallas, TX) solution (1:100) for 3 hours at RT with moderate rocking. After washing the wells, cells were incubated with goat anti-mouse secondary antibody (IRDye 800CW) (1:200) in 0.1% Tween 20 for 1 hour at RT in the dark. After the incubation was complete the wells were washed 3 times using PBST and once with PBS and further imaged using LICOR Odyssey infrared imaging system.

The osteogenicity was monitored by quantifying relative protein expression levels of Runx2 with an ICW experiment. Briefly, after reading the Alamar blue levels, the media with alamar blue was discarded from each well, then the cells were washed with warm PBS and fixed with 4% paraformaldehyde solution for 15 minutes followed by washing and permeabilizing with 0.1% Triton X-100 in PBS 5 times each. Then the plate was blocked using Odyssey blocking buffer for 1 hour and incubated with Runx2 primary mouse antibody (Santa Cruz Biotechnology, Dallas, TX) solution (1:100) for 3 hours at RT with moderate rocking. Well H12 in the 96 well plate (Fig. 1B) was assigned as a control well and incubated with blocking buffer without primary antibody and further used to correct background. After washing the wells, cells were incubated with goat anti-mouse secondary antibody (IR 800CW) (1:200) and 0.1% Tween 20 for 1 hr at RT in dark. After the incubation was complete the wells were washed 3 times using PBST and once with PBS and further imaged using LICOR Odyssey infrared imaging system.