PAG1 Antibody / Cbp / Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (FY13067)

Immunofluorescent staining of PAG1 using anti-PAG1 antibody (green). PAG1 was detected in an immunocytochemical section of U2OS 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-PAG1 antibody overnight at 4oC. DyLight 488 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 PAG1 using anti-PAG1 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human HEL whole cell lysates, Lane 2: human THP-1 whole cell lysates, Lane 3: rat brain tissue lysates, Lane 4: rat spleen 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-PAG1 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 an ECL Plus Western Blotting Substrate. Western blot analysis of PAG1 (Cbp) expression in human and rodent samples. Although PAG1 has a predicted molecular weight of ~47 kDa, it consistently migrates at ~68-72 kDa on SDS-PAGE due to its dual palmitoylation and extensive tyrosine phosphorylation. Human lysates display a characteristic doublet, corresponding to differently phosphorylated forms of PAG1, while rodent tissues show a single dominant species at similar apparent size.

Flow Cytometry analysis of RT4 cells using anti-PAG1 antibody. Overlay histogram showing RT4 cells stained with (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-PAG1 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.