POMT1 Antibody / Protein O-mannosyl-transferase 1 (FY13225)

Flow Cytometry analysis of MCF-7 cells using anti-POMT1 antibody. Overlay histogram showing MCF-7 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-POMT1 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 POMT1 using anti-POMT1 antibody. Lane 1: human 293T whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human MCF-7 whole cell lysates, Lane 4: human Hacat whole cell lysates, Lane 5: rat 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-POMT1 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. Western blot detection of POMT1 shows a band at ~70-75 kDa across multiple lysates. Although the predicted mass is ~85 kDa, POMT1 commonly migrates lower on SDS-PAGE, consistent with maturation/processing of the luminal N-terminus and isoform- or proteolysis-related heterogeneity typical of ER membrane glycosyltransferases.