Immunocytochemistry/Immunofluorescence analysis using Mouse Anti-Nitrotryptophan Monoclonal Antibody, Clone 2D12. Tissue: Embryonic kidney epithelial cell line (HEK293). Species: Human. Fixation: 5% Formaldehyde for 5 min. Primary Antibody: Mouse Anti-Nitrotryptophan 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) DAPI (blue) nuclear stain. (B) Phalloidin Alex Fluor 633 F-Actin stain. (C) Nitrotryptophan Antibody. (D) Composite. Courtesy of: Dr. Robert Burke, University of Victoria.
Western Blot analysis of 6-Nitrotryptophan-BSA Conjugate showing detection of 67 kDa Nitrotryptophan protein using Mouse Anti-Nitrotryptophan Monoclonal Antibody, Clone 2D12. Lane 1: Molecular Weight Ladder (MW). Lane 2: BSA (0.5 ug). Lane 3: BSA (1 ug). Lane 4: 6-Nitrotryptophan-BSA (0.5 ug). Lane 5: 6-Nitrotryptophan-BSA (1 ug). Lane 6: 7-Ketocholesterol-BSA (0.5 ug). Lane 7: 7-Ketocholesterol-BSA (1 ug). Block: 5% Skim Milk in TBST. Primary Antibody: Mouse Anti-Nitrotryptophan 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: Luminol for 1 min. Predicted/Observed Size: 67 kDa.
Flow Cytometry analysis using Mouse Anti-Nitrotryptophan Monoclonal Antibody, Clone 2D12. Tissue: Neuroblastoma cells (SH-SY5Y). Species: Human. Fixation: 90% Methanol. Primary Antibody: Mouse Anti-Nitrotryptophan 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 uM H2O2 for 24 hours.
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