Tuesday, February 14, 2012
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.)
Monoclonal Anti-Desmin Antibody from Sigma-Aldrich, used in Western Blotting
The Good
Our results were quite reproducible.
The Bad
We had to load large amounts of protein when using whole tissue extracts.
The Bottom Line
Consistent, reproducible results.