MICAL3 Antibody / Molecule interacting with CasL protein 3 (FY12787)

Flow Cytometry analysis of U251 cells using anti-MICAL3 antibody. Overlay histogram showing U251 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-MICAL3 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 (Red line) was also used as a control.

Western blot analysis of MICAL3 using anti-MICAL3 antibody. Lane 1: human U251 whole cell lysates, Lane 2: human SiHa whole cell lysates, Lane 3: human whole cell lysates, Lane 4: human Hacat whole cell lysates, Lane 5: rat testis tissue lysates, Lane 6: mouse testis tissue 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-MICAL3 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. The signal was developed using enhanced chemiluminescent. Multiple bands are detected between ~200 and 250 kDa with an occasional higher species, consistent with reported post-translationally modified forms and partially SDS-resistant complexes of the large MICAL3 protein (predicted ~224 kDa).