NEK7 Antibody / NIMA related kinase 7 (FY12050)

Immunofluorescent staining of NEK7 using anti-NEK7 antibody (green) and anti-Beta Tubulin antibody (red). NEK7 was detected in an immunocytochemical section of SiHa 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-NEK7 antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG and Cy3 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of NEK7 using anti-NEK7 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 Hela whole cell lysates, Lane 3: human Jurkat whole cell lysates, Lane 4: human SIHA whole cell lysates, Lane 5: rat heart tissue lysates, Lane 6: mouse lung tissue lysates, Lane 7: mouse heart 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-NEK7 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. A specific band was detected for NEK7 at approximately 32 kDa.

Immunoprecipitating NEK7 in Jurkat whole cell lysate. Western blot analysis of NEK7 using anti-NEK7 antibody. Lane 1: Jurkat whole cell lysates (30ug), Lane 2: Rabbit control IgG instead of anti-NEK7 antibody in Jurkat whole cell lysate, Lane 3: anti-NEK7 antibody (2ug) + Jurkat whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-NEK7 antibody at a dilution of 0.5 ug/ml and probed with a goat anti-rabbit IgG-HRP secondary antibody. The signal is developed using ECL Plus Western Blotting Substrate. A specific band was detected for NEK7 at approximately 32 kDa. The expected band size for NEK7 is at 35 kDa.

Flow Cytometry analysis of 293T cells using anti-NEK7 antibody. Overlay histogram showing 293T 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-NEK7 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.