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Monoclonal anti-Human Dysferlin

cat# RDI-DYSFERLabm     $500.00/vial

Specificity:* recognizes Human, mouse, rat, rabbit, hamster and dog muscle dysferlin (not chicken). Other species not tested. Also present inmany non- mucle tissues. Severly reduced of labeling in the SJL mouse.

Presentation: 1 ml lyophilized supernatant with 15mM sodium azide added.

Clone: HAM1/7B6

Isotype: mIgG1k

Antigen: synthetic peptide containing amino acids 1999-2016 of the human dysferlin moleculae (within exon 53)

Hybridoma: mouse myeloma (X63.Ag8.653)XCD1

USES: -frozen section (unfixed-see protocol) or : optimum fixative acetone/methanol (1:1) 4 minutes at 25 DEg C. approx 1:20-1:40

-Paraffin sections using high temp antigen release

-typical dilutions:1:20-1:40, 60 minute incubation at 25 DEG C. ABC detection

-western blot:not evaluated

Stain: membrane staining of muscle fibres (also shows slight cytoplasmic localization in a fibre-type mosaic)

Controls: normal human skeletal muscle

Reconstitution: 1 ml distilled water. Let set at least 30 minutes. Tighten cap and mix gently.

Storage: Store lyophilized material at 4 DEG C. Store reconstituted material at 4 DEG C for immediate use. For long term use, recommend aliquoting and store at -20 DEG C. Avoid frequent freeze thaw cycles.

Background:Dysferlin is the protein product of the 2p13 gene that is defective in patients with Limb-Girdle Muscular Dystrophy type 2B (LGMD2B) and Miyoshi Myopathy (MM). Dysferlin is normally localized to the muscle plasma membrane. In patients with LGMD2B and MM, immunoreactivity to dysferlin is severly reduced or lost, depending on the type of mutation. Patients with other neuromuscular conditions demonstrate normal labelling patterns, so this antibody may be if use for the characterization of LGMD2B and MM. Labelling with an antibody to beta-spectrin, to monitor membrane integrity is an essential immunohistochemical control.




Protocol for UNFIXED Frozen Sections:

1. Freeze muscle blocks in isopentane chilled in liquid   nitrogen

2. Cut 4-10 micron sections and air dry on slides coated with   section adhesive.

3. Slides may be stored below -70 DEG C wrapped in cling film   until required. If stored sections are used, allow sections   to equilibrate to 25 DEG C before unwrapping and proceeding.

4. Apply a 50ul of dilute dprimary antibody to section   (unfixed). Incubate for 1 hour at 25-37 DEG C

5. Wash sections 3 X 10 minutes in phosphate buffered saline

6. Apply a 50ul aliquot of labelled secondary antibody.   Incubate for 1 hour at 25 DEg C.

7. Wash sections 3 X 10 minutes in phosphate buffered saline

8. Mount fluorescent sections in aqueous mountant or visualize   peroxidase label (eg by exposure to freshly prepared 0.05%   w/v diaminobenzene in PBS containing 0.1% w/v hydrogen   peroxide.). Dehydrate, clear and mount peroxidase labelled   sections for permanent preparations.

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