Editor's Note: All antibody reviews appear in protocol format.
Primary Antibody: Mouse Ascites Fluid Anti-Desmin Monoclonal Antibody, Unconjugated, Clone DE-U-10
Antigen/Epitope: Murine desmin
Supplier and catalog number: Sigma-Aldrich D1033
Sample: Murine skeletal muscle, 100 ug total protein (as determined by BCA Protein Assay (Thermo Scientific, Pierce Protein Research Products, Rockford, IL)
Secondary Antibody: Goat Anti-Mouse IgG (H+L)-HRP conjugate
Supplier and catalog number: Bio-Rad 172-1011
Block: 5% nonfat milk in TBS-Tween20
Incubation time: 1 hr
Incubation temperature: room temperature
Primary Antibody Incubation
Dilution used: 1/150
Diluent composition: TBS-Tween 20
Incubation time: overnight
Incubation temperature: 4°C
Secondary Antibody Incubation
Dilution used: 1/20000
Diluent composition: TBS-Tween20 + 1% bovine serum albumin (BSA)
Incubation time: 1 hr
Incubation temperature: room temperature
Detection Method: Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Pierce Protein Research Products, Rockford, IL) + film
Results Summary: In vivo, we analyzed regenerating skeletal muscle, otherwise desmin expression was very low; in vitro desmin is normally expressed by myogenic cell lines / primary cultures. Our results were quite reproducible but we had to load large amounts of protein when using whole tissue extracts.
Additional notes/tips: Apparent molecular weight: 53 Kda, after blotting onto nitrocellulose mebrane.
(Note: The image at the top of this page is from Sigma-Aldrich.)