Anti-Dityrosine Antibody [10A6] (A304720)

Flow cytometry analysis of human neuroblastoma cells (SH-SY5Y), fixed in 90% methanol, using Anti-Dityrosine Antibody [10A6] (A304720) at 1:50 for 30 minutes on ice. The secondary antibody used was Goat Anti-Mouse: PE at 1:100 for 20 minutes at room temperature. Isotype Control: Non Specific IgG. Cells were subject to oxidative stress by treating with 250 µM H2O2 for 24 hours.

Western blot analysis of human Cervical cancer cell line (HeLa) lysate showing detection of Dityrosine protein using Anti-Dityrosine Antibody [10A6] (A304720) at 1:1,000 for 2 hours at room temperature. Lane 1: Molecular Weight Ladder (MW). Lane 2: HeLa cell lysate. Lane 3: H2O2 treated HeLa cell lysate. Load: 12 µg. Block: 5% Skim Milk in TBST. The secondary antibody used was Goat Anti-Mouse IgG: HRP at 1:2000 for 60 minutes at room temperature. Color Development: ECL solution for 5 minutes in room temperature.

Western blot analysis of Dityrosine-BSA Conjugate showing detection of 67 kDa Dityrosine protein using Anti-Dityrosine Antibody [10A6] (A304720) at 1:1,000 for 2 hours at room temperature. Lane 1: Molecular Weight Ladder (MW). Lane 2: BSA. Lane 3: 3,5-Dibromotyrosine-BSA. Lane 4: Dityrosine-BSA. Lane 5: Bromotyrosine-BSA. Lane 6: 7-ketocholesterol-BSA. Load: 1 µg. Block: 5% Skim Milk in TBST. The secondary antibody used was Goat Anti-Mouse IgG: HRP at 1:2000 for 60 minutes at room temperature. Color Development: ECL solution for 5 minutes in room temperature. Predicted/Observed Size: 67 kDa.

Immunocytochemistry/Immunofluorescence analysis of human embryonic kidney epithelial cell line (HEK293), fixed in 5% formaldehyde for 5 minutes, using Anti-Dityrosine Antibody [10A6] (A304720), at 1:50 for 30-60 min at room temperature. The secondary antibody used was Goat Anti-Mouse Alexa Fluor 488 at 1:1500 for 30-60 min at room temperature. Counterstain: Phalloidin Alexa Fluor 633 F-Actin stain; DAPI (blue) nuclear stain at 1:250, 1:50,000 for 30-60 min at room temperature. Localization: Cytoplasmic. Magnification: 20X (2X Zoom). (A,C,E,G) Untreated. (B,D,F,H) Cells cultured overnight with 50 µM H2O2. (A,B) DAPI (blue) nuclear stain. (C,D) Phalloidin Alexa Fluor 633 F-Actin stain. (E,F) Dityrosine Antibody. (G,H) Composite.