PRAG1 Antibody / Pragmin / PEAK1-related kinase-activating pseudokinase 1 (FY12864)

Immunofluorescent staining of PRAG1 using anti-PRAG1 antibody (red). PRAG1 was detected in an immunocytochemical section of human HELA cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 ug/ml rabbit anti-PRAG1 antibody overnight at 4oC. Cy3 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. The section was counterstained with DAPI nuclear stain (blue). Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of PRAG1 using anti-PRAG1 antibody. Lane 1: human whole cell lysates, Lane 2: human HepG2 whole cell lysates, Lane 3: human Caco-2 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-PRAG1 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. PRAG1 western blot of human cell lines shows a predominant band near ~200 kDa. Although the theoretical mass is ~150 kDa, PRAG1 commonly migrates higher due to extensive phosphorylation, glycosylation, and its proline-rich structure. The ~200 kDa band therefore corresponds to full-length, post-translationally modified PRAG1.

Flow Cytometry analysis of HepG2 cells using anti-PRAG1 antibody. Overlay histogram showing HepG2 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-PRAG1 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.