PDZK1 Antibody / PDZ domain-containing 1 / NHERF3 (FY13097)

Immunohistochemical staining of PDZK1 using anti-PDZK1 antibody. PDZK1 was detected in a paraffin-embedded section of human liver tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-PDZK1 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Western blot analysis of PDZK1 using anti-PDZK1 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human 293T whole cell lysates, Lane 2: human Caco-2 whole cell lysates, Lane 3: human HepG2 whole cell lysates, Lane 4: human MCF-7 whole cell lysates, Lane 5: rat kidney tissue lysates, Lane 6: rat liver tissue lysates, Lane 7: mouse kidney tissue lysates, Lane 8: mouse liver tissue lysates. Lane 8: mouse brain 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-PDZK1 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. Although PDZK1 is ~57 kDa by sequence, the antibody detects a predominant species at ~70 kDa in most samples. This upward shift is well known for PDZK1 and reflects its acidic, multi-PDZ architecture and phosphorylation, which slow SDS–PAGE mobility. In kidney tissue, PDZK1 migrates slightly faster, consistent with reduced phosphorylation and/or expression of shorter renal isoforms.

Flow Cytometry analysis of RM-1 cells using anti-PDZK1 antibody. Overlay histogram showing RM-1 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-PDZK1 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.

Flow Cytometry analysis of CACO-2 cells using anti-PDZK1 antibody. Overlay histogram showing CACO-2 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-PDZK1 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.