Purified Mouse Anti-MyoD Monoclonal Antibody: BC1-C2H8
Product Number: M6000PU
Size: 0.1 mg
Concentration: 0.25 mg/ml
Immunogen: 14 amino acid peptide whose sequence corresponds to a region that is specific to the MyoD protein. This region is distal (away) from the homologous helix-loop-helix region of the MyoD protein which is shared by other members of the myogenic bHLH transcription factors.
Isotype: Mouse IgG1
Reactivity: Human, mouse and rat myogenic cell lines and tissues with ongoing myogenesis.
Staining of Rh30 rhabdomyosarcoma cells at 5ug/ml is shown. Secondary antibody; FITC conjugated anti mouse Ig.
Storage Buffer: Phosphate buffered saline containing 0.05% sodium azide.
MyoD is a myogenic determination basic helix loop helix protein that comprises 318 amino acids. The human homolog is known as Myf 3. This transcription factor is expressed in myogenic cells and developing myogenic tissues. MyoD belongs to the family of myogenic transcription factors which include myf 5, myogenin (myf4) and MRF4 (myf6 or herculin) that are involved in programming various stages of skeletal muscle development. It has been shown that forced expression of MyoD (or related members) in non-muscle cells either converts the cells to myogenic cells or transcriptionally activates a set of otherwise silent muscle-specific genes in these cells. Members of the MyoD family play distinct roles during muscle development; MyoD and Myf-5 act early during myogenesis. Inactivation of MyoD results in the up-regulation of myf5 and similarly inactivation of myf5 results in the up-regulation of MyoD. Inactivation of both MyoD and Myf 5 results birth of mice that lack muscle. Myogenin and MRF4 act later in myogenesis and are associated with muscle maturation. Members of the MyoD1 family, in particular MyoD, have been useful as markers for identifying cells of the myogenic lineage. This has been particularly useful for distinguishing poorly differentiated rhabdomyosarcoma (tumor of the skeletal muscle lineage) from other non muscle tumors. Thus, antibodies to MyoD have been particularly useful for distinguishing rhabdomyosarcomas from other small round cell tumors or poorly differentiated tumors that have with minimum signs of lineage specific differentiation.
The antibody is routinely tested on myogenic cell lines by immunostaining on and by western blotting. Suggested positive control cell lines include human rhabdomyosarcoma cell lines such as Rh30 (ATCC CRL-2061) & RD (ATCC CCL-136), mouse myogenic cell lines such as C2C12 (ATCC CRL 1772) and rat myogenic cell lines such as L6 (ATCC CRL-1458).
Preparation and Storage
The monoclonal antibody may be supplied as ascites fluid or is purified from tissue culture supernatant with Protein G column chromatography.
Store undiluted at 4° C or aliquot and store at -20C for long term storage. Repeated freeze thaw cycles should be avoided.
Immunohistochemistry-The antibody may be used on cryosections of embryonic muscle tissues and rhabdomyosarcoma tissues. For such applications, it is recommended that the cryosections are fixed in 1-2% paraformaldehyde for 30 mins followed by permeabilization in 0.25% Triton X 100 in PBS for 30 mins. After blocking of non-specific binding with 1% BSA in PBS, incubate with primary antibody diluted at 3-5ug/ml in 1%BSA in PBS. Please note that this reagent is sold strictly for use as a research reagent.
Western blotting- Use the antibody at 2-3 ug/ml in Tris buffered Saline with 0.05% Tween 20 and 5% non-fat dry milk or similar diluents.
Rh30 human rhabdomyosarcoma cells (ATCC CRL-2061) or RD human rhabdomyosarcoma cells (ATCC CCL-136) are suggested as positive control cell lines for these applications. The antibody reacts with doublet bands of approximate molecular size 45kD. Cell lines that are suggested as negative controls include Ewing’s sarcoma cells RD-ES (ATCC HBT-166) and Burkitt’s lymphoma cells EB2 (ATCC HTB-61).
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Caution: The product contains 0.05% sodium azide. Sodium azide produces highly toxic hydrazoic acid under acidic conditions. It is therefore recommended to dilute azide compounds in running water before discarding to prevent the accumulation of potentially explosive deposits in the plumbing.
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