Anti-Dystrophin Antibody

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

Epitope: mid-rod

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.

Research Category
Metabolism

Research Sub Category
Muscle Physiology

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

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.

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

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

Control
POSITIVE CONTROL: Snap frozen normal human or rat striated muscle.

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