BCKDH E2 Antibody / Dihydrolipoamide branched chain transacylase E2 / DBT (FY13381)

Immunofluorescent staining of BCKDH E2/DBT using anti-BCKDH E2 antibody (green) and anti-Beta Tubulin antibody (red). BCKDH E2/DBT 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-BCKDH E2 antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG and DyLight 550 Conjugated Goat Anti-Mouse IgG were 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 BCKDH E2/DBT using anti-BCKDH E2 antibody. Lane 1: human SiHa whole cell lysates, Lane 2: human whole cell lysates, Lane 3: human Hacat whole cell lysates, Lane 4: human Jurkat whole cell lysates, Lane 5: rat stomach tissue lysates, Lane 6: rat kidney tissue lysates, Lane 7: mouse stomach tissue lysates, Lane 8: mouse kidney 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-BCKDH E2 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. BCKDH E2 (DBT) antibody detects a single strong band just below the 50 kDa marker in human cell and tissue lysates, slightly faster than the 53.5 kDa predicted mass but consistent with the processed 52 kDa mitochondrial autoantigen form of DBT reported in the literature and typical SDS-PAGE migration behavior of mitochondrial enzymes.

Immunoprecipitating BCKDH E2/DBT in whole cell lysate. Western blot analysis of BCKDH E2/DBT using anti-BCKDH E2 antibody. Lane 1: whole cell lysates (30ug), Lane 2: Rabbit control IgG instead of anti-BCKDH E2 antibody in whole cell lysate, Lane 3: anti-BCKDH E2 antibody (2ug) + whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-BCKDH E2 antibody at a dilution of 0.5 ug/ml and probed with a mouse anti-rabbit IgG-HRP secondary antibody (Light Chain). The signal is developed using ECL Plus Western Blotting Substrate. BCKDH E2 (DBT) antibody detects a single strong band just below the 50 kDa marker in human cell and tissue lysates, slightly faster than the 53.5 kDa predicted mass but consistent with the processed 52 kDa mitochondrial autoantigen form of DBT reported in the literature and typical SDS-PAGE migration behavior of mitochondrial enzymes.

Flow Cytometry analysis of human SiHa cells using anti-BCKDH E2 antibody. Overlay histogram showing SiHa 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-BCKDH E2 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.