TBKBP1 Antibody / TANK-binding kinase 1-binding protein 1 (FY13290)

Immunofluorescent staining of TBKBP1 using anti-TBKBP1 antibody (green) and anti-Beta Tubulin antibody (red). TBKBP1 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-TBKBP1 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 TBKBP1 using anti-TBKBP1 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 Caco-2 whole cell lysates, Lane 3: human 293T whole cell lysates, Lane 4: human SH-SY5Y whole cell lysates, Lane 5: rat lung tissue lysates, Lane 6: rat brain tissue lysates, Lane 7: mouse lung 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-TBKBP1 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 predominant band is detected at an approximately 78 kDa in all samples, running above the predicted ~68 kDa size but consistent with the higher apparent molecular weight expected for a coiled-coil adaptor protein with multiple post translational modifications. Some lysates also display a weaker band between ~65 and 70 kDa, likely representing a less modified or partially processed form of TBKBP1.

Flow Cytometry analysis of SH-SY5Y cells using anti-TBKBP1 antibody. Overlay histogram showing SH-SY5Y 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-TBKBP1 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.