Fibroblast lysates were obtained by sonication (details under MUT activity assay). Crude cell lysates (25 μg of protein per sample) were mixed with 2× Laemmli Sample Buffer (Bio-Rad, Hercules, CA, USA) and heated to 96°C for 6 min. Proteins were separated by 10% SDS-PAGE, transferred to a Protran BA85 nitrocellulose membrane (Whatman, GE Healthcare), blocked at room temperature for 1 hr with buffer A (5% skimmed milk, 1.2% w/v Tris-base, 9% w/v NaCl, 0.2% Tween 20, pH 7.6), incubated with polyclonal mouse anti-human MUT (Abcam, Cambridge, UK) (1:1,000 in buffer A), or β-actin (Sigma–Aldrich, Buchs SG, Switzerland) (1:4,000 in buffer A) overnight, detected by incubation with goat anti-mouse antibody coupled to horseradish peroxidase (Santa Cruz, Biotechnology, Dallas, TX, USA) (1:5,000 in buffer A) for 1 hr, and visualized with ECL Western Blotting Detection Reagents (GE Healthcare) on a Gel Logic 6000 Pro (Carestream, Gland, Switzerland).
Goat anti mouse antibody coupled to horseradish peroxidase
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Goat anti-mouse antibody coupled to horseradish peroxidase is a laboratory reagent used as a secondary antibody in various immunoassays and immunohistochemical techniques. The antibody is produced in goats and is specific to mouse primary antibodies, while the horseradish peroxidase enzyme serves as a label for detection and visualization purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ecl western blotting detection reagent, by GE Healthcare (2 mentions)
Polyclonal mouse anti human mut, by Abcam (2 mentions)
Gel logic 6000 pro, by Carestream (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
β actin, by Merck Group (2 mentions)
Protran ba85 nitrocellulose membrane, by Cytiva (2 mentions)
2 protocols using goat anti mouse antibody coupled to horseradish peroxidase
1
Fibroblast MUT Protein Detection
2
Quantification of Mutant Methylmalonyl CoA Mutase
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Fibroblast lysates were obtained by sonication (details under MUT activity assay). Crude cell lysates (25 μg of protein per sample) were mixed with 2× Laemmli Sample Buffer (Bio‐Rad, Hercules, CA, USA) and heated to 96°C for 6 min. Proteins were separated by 10% SDS‐PAGE, transferred to a Protran BA85 nitrocellulose membrane (Whatman, GE Healthcare), blocked at room temperature for 1 hr with buffer A (5% skimmed milk, 1.2% w/v Tris‐base, 9% w/v NaCl, 0.2% Tween 20, pH 7.6), incubated with polyclonal mouse anti‐human MUT (Abcam, Cambridge, UK) (1:1,000 in buffer A), or β‐actin (Sigma–Aldrich, Buchs SG, Switzerland) (1:4,000 in buffer A) overnight, detected by incubation with goat anti‐mouse antibody coupled to horseradish peroxidase (Santa Cruz, Biotechnology, Dallas, TX, USA) (1:5,000 in buffer A) for 1 hr, and visualized with ECL Western Blotting Detection Reagents (GE Healthcare) on a Gel Logic 6000 Pro (Carestream, Gland, Switzerland).
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