Immunocytochemistry/Immunofluorescence analysis using Mouse Anti-7-Ketocholesterol Monoclonal Antibody, Clone 7E1. Tissue: Embryonic kidney epithelial cell line (HEK293). Species: Human. Fixation: 5% Formaldehyde for 5 min. Primary Antibody: Mouse Anti-7-Ketocholesterol 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) 7-Ketocholesterol Antibody. (G,H) Composite. Courtesy of: Dr. Robert Burke, University of Victoria.
Western Blot analysis of 7-Ketocholesterol-BSA Conjugate showing detection of 67 kDa 7-Ketocholesterol protein using Mouse Anti-7-Ketocholesterol Monoclonal Antibody, Clone 7E1. Lane 1: Molecular Weight Ladder (MW). Lane 2: BSA (0.5 ug). Lane 3: BSA (2.0 ug). Lane 4: 7-ketocholesterol-BSA (0.5 ug). Lane 5: 7-ketocholesterol-BSA (2.0 ug). Block: 5% Skim Milk in TBST. Primary Antibody: Mouse Anti-7-Ketocholesterol 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.
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