ITPKC Antibody / Inositol-trisphosphate 3-kinase C (FY12625)

Flow Cytometry analysis of Caco-2 cells using anti-ITPKC antibody. Overlay histogram showing Caco-2 cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-ITPKC antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Western blot analysis of ITPKC using anti-ITPKC antibody. Lane 1: human RT4 whole cell lysates, Lane 2: human Caco-2 whole cell lysates, Lane 3: human whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-ITPKC antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. Western blot probed with anti-ITPKC shows a dominant band at ~90 kDa, higher than the predicted ~75 kDa, consistent with phosphorylated forms of ITPKC. A faint band near 100 kDa likely represents a hyperphosphorylated or sumoylated variant of the kinase.