Analysis Note

ControlPOSITIVE CONTROL: Snap frozen normal human or rat striated muscle.

Application

Research Sub CategoryMuscle Physiology

Research CategoryMetabolism

This Anti-Dystrophin Antibody, mid-rod, clone 6D3 is validated for use in WB, IH for the detection of Dystrophin.

Immunohistochemistry: (fresh frozen, unfixed tissue only): use

undiluted - 1:20. Not recommended for use on paraffin embedded tissue.

EM Gold (Light fixation with 2% formaldehyde + 0.001% glutaraldehyde for 1 hour. 2.3M sucrose used as cryoprotectant.): use undiluted. 90 minute incubation at 25°C.

Western blotting: use 1:100-1:250.

Optimal working dilutions must be determined by the end user.

Protocol for Immunohistochemical use of MAB1692

1) Freeze muscle blocks in isopentane chilled in liquid nitrogen.

2) Cut 4 µm to 10 µm sections and air dry on slides coated with 0.5% gelatin containing 0.05% chrome alum.

3) Slides may be stored at -70 °C wrapped in cling film until required. If stored sections are used, allow sections to equilibrate to room temperature before unwrapping and proceeding.

4) Apply a 50 µL aliquot of primary antibody to sections (unfixed). Incubate for 1 hour at room temperature or 37°C.

5) Wash sections 3 x 10 minutes in phosphate buffered saline.

6) Apply a 50 µL aliquot of labeled second antibody. Incubate for 60 minutes at 25°C.

7) Wash sections 3 x 10 minutes in phosphate buffered saline.

8) Mount fluorescent sections in aqueous mounting media or visualize peroxidase label (DAB). Dehydrate, clean and mount peroxidase labeled sections for permanent preparations.

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Immunogen

Epitope: mid-rod

Bacterial fusion protein (Cell (1987) 51:919-928).

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Physical form

Culture supernatant, liquid in PBS with 1% BSA, containing 15 mM sodium azide.

Specificity

Mid rod domain (between amino acids 1181 and 1388) of human dystrophin. Also reacts with skeletal, cardiac and smooth muscle dystrophin from normal mouse, rat, rabbit and dog. Other animal species have not been tested. Reacts on blots with the brain isoform. No reactivity with mdx mouse tissue or DMD/BMD patients who have a gene deletion which removes the antibody binding site. Does not react with chicken dystrophin.

STAINING PATTERN:Light microscopy: continuous rim of labeling at the periphery of muscle fibers.

E.M. gold: close to the cytoplasmic face of the plasma membrane.

Western blotting: strong double bands at approximately 400 kD plus metabolites of lower molecular mass.

Storage and Stability

Maintain at -20°C for up to one year in convenient undiluted aliquots. Avoid repeated freeze/thaw cycles.