Immunocytochemistry/Immunofluorescence analysis using Mouse Anti-Acrolein Monoclonal Antibody, Clone 10A10. Tissue: Embryonic kidney epithelial cell line (HEK293). Species: Human. Fixation: 5% Formaldehyde for 5 min. Primary Antibody: Mouse Anti-Acrolein 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. Magnification: 20X (2X Zoom). (A,C,E,G) - Untreated. (B,D,F,H) - Cells cultured overnight with 50 uM H2O2. (A,B) DAPI (blue) nuclear stain. (C,D) Phalloidin Alex Fluor 633 F-Actin stain. (E,F) Acrolein Antibody. (G,H) Composite. Courtesy of: Dr. Robert Burke, University of Victoria.
Western Blot analysis of Acrolein-BSA Conjugate showing detection of 67 kDa Acrolein protein using Mouse Anti-Acrolein Monoclonal Antibody, Clone 10A10. Lane 1: Molecular Weight Ladder (MW). Lane 2: AcroleinBSA (0.5 ug). Lane 3: AcroleinBSA (2.0 ug). Lane 4: BSA (0.5 ug). Lane 5: BSA (2.0 ug). Block: 5% Skim Milk in TBST. Primary Antibody: Mouse Anti-Acrolein 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-Acrolein Monoclonal Antibody, Clone 10A10. Tissue: Neuroblastoma cells (SH-SY5Y). Species: Human. Fixation: 90% Methanol. Primary Antibody: Mouse Anti-Acrolein 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.
Western Blot analysis of Human Cervical cancer cell line (HeLa) lysate showing detection of Acrolein protein using Mouse Anti-Acrolein Monoclonal Antibody, Clone 10A10. Lane 1: Molecular Weight Ladder (MW). Lane 2: HeLa cell lysate. Lane 3: H2O2 treated HeLa cell lysate. Load: 12 ug. Block: 5% Skim Milk in TBST. Primary Antibody: Mouse Anti-Acrolein 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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