Application Notes: A 1:1000 dilution of SMC-521 was sufficient for detection of dityrosine in 1 µg of Dityrosine conjugated to BSA by ECL immunoblot analysis using Goat Anti-Mouse IgG:HRP as the secondary Antibody
Immunocytochemistry/Immunofluorescence analysis using Mouse Anti-Dityrosine Monoclonal Antibody, Clone 10A6. Tissue: Embryonic kidney epithelial cell line (HEK293). Species: Human. Fixation: 5% Formaldehyde for 5 min. Primary Antibody: Mouse Anti-Dityrosine Monoclonal Antibody at 1:50 for 30-60 min at RT. Secondary Antibody: Goat Anti-Mouse Alexa Fluor 488 at 1:1500 for 30-60 min at RT. Counterstain: Phalloidin Alexa Fluor 633 F-Actin stain, DAPI (blue) nuclear stain at 1:250, 1:50000 for 30-60 min at RT. 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.
Western Blot analysis of Dityrosine-BSA Conjugate showing detection of 67 kDa Dityrosine protein using Mouse Anti-Dityrosine Monoclonal Antibody, Clone 10A6. 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. Primary Antibody: Mouse Anti-Dityrosine Monoclonal Antibody at 1:1000 for 2 hours at RT. Secondary Antibody: Goat Anti-Mouse IgG: HRP at 1:2000 for 60 min at RT. Color Development: ECL solution for 5 min in RT. Predicted/Observed Size: 67 kDa.
Flow Cytometry analysis using Mouse Anti-Dityrosine Monoclonal Antibody, Clone 10A6. Tissue: Neuroblastoma cells (SH-SY5Y). Species: Human. Fixation: 90% Methanol. Primary Antibody: Mouse Anti-Dityrosine Monoclonal Antibody at 1:50 for 30 min on ice. Secondary Antibody: Goat Anti-Mouse: PE at 1:100 for 20 min at RT. 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 Mouse Anti-Dityrosine Monoclonal Antibody, Clone 10A6. 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. Primary Antibody: Mouse Anti-Dityrosine Monoclonal Antibody at 1:1000 for 2 hours at RT. Secondary Antibody: Goat Anti-Mouse IgG: HRP at 1:2000 for 60 min at RT. Color Development: ECL solution for 5 min in RT.
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