Mothers Against Decapntaplegic

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans.  Approximately 120 mb euchromatic portion of the fly genome ha been sequenced using a shotgun sequencing approach.  The entire fly genome encodes for approximately 13,600 genes with vast functional diversity.  The dorsal ventral patterning in vertebrate embryos is regulated by members of TGF-beta family of growth and differentiation factors.  The receptors for ligands in theTGF-beta superfamily are kinases but not much is known about their down stream components.  In drosophila the decapentaplegic protein (dpp), a homolog of vertebrate BMP2 and BMP4 is involved in dorsal ventral axial patterning. One of the down stream component of dpp signaling is MAD that codes a unique predicted cytoplasmic with no identifiable functional domain.  MAD is a member of a novel, highly conserved protein family.  The drosophila homoplog of Mad in xenopus (XMAD) exhibits activities consistent with a role in BMP signaling, it rescues phenotypes generated by a signaling-defective, dominant-negative, BMP-2/4 receptor.  XMad protein induces ventral mesoderm when over expressed in isolated animal caps and it ventralizes embryos (1).  Homozygous Mad mutant animals exhibit defects in midgut morphogenesis, imaginal disk development and embryonic dorsal-ventral patterning that are very reminiscent of dpp mutant phenotypes (2).

The protein Mad is a 455 amino acid (MW 56kDa) cytosolic protein expressed during early developmental stages of fly.  The mammalian counter parts of Mad are several isoforms of SMAD (SMAD1-8) acting as a down stream transcriptional activators.  SMAD2 and SMAD3 are phosphorylated as a result of the canonical cascade through ligand binding and receptor kinase activation. These phosphorylated SMADs (pSMAD) associate with SMAD4, a co-SMAD, and transcriptionally activate TGFbeta-mediated genes.  In drosophila Mad is phosphorylation is noted during late embryonic stage 5 (E5) and early E6 stages of development.  We have also made a phospho-specific Mad antibody against synthetic peptide corresponding to amino acid 446-455 containing phosphoserine at position 453 and 455.  Mad also regulates synaptic development in conjunction with Thickvein.  During synaptic development Mad undergoes phosphorylation in regions facing the presynaptic active zones of neurotransmitter release (3). 

The Mad-selective antibodies were generated against synthetic peptides form unique regions on the C-terminal end of protein.  The Mad peptide was post-synthetically modified to achieve the desired antigenicity before injecting in to rabbits to obtained antibodies. The antibodies were isolated on an immobilized antigen based affinity matrix before stabilizing them in antibody stabilization buffer.  These antibodies label Mad protein as a single 56kDa band in PC-Mad samples.  FabGennix Inc. provides Western blot positive control samples for Mad in ready-to-use buffer for SDS-PAGE and western blotting applications.  Mad positive control appears as a diffuse band of 56kDa.   FabGennix International Inc., has also produced a phosphor-Mad antibody (Cat # PMad-140AP).  Limited quantities of antigenic blocking peptides for these antibodies are also available (Inquire before placing orders).  For a complete listing of all Drosophila related antibodies, visit our website at www.FabGennix.com.   FabGennix International Inc., will conjugate and couple these antibodies to fluorescent probes and secondary enzymes at a nominal price.

Immunogen:            Mad antibodies are developed against conserved antigenic peptide sequences corresponding to C-terminal end of the protein, the peptide was covalently modified to achieve desired antigenicity before used as antigen.

Concentration:         Antibodies Mad-101AP has immobilized antigen based affinity chromatography purified immunoglobulin concentration of 0.5-0.73mg/ml in antibody stabilization buffer.   

Applications:           ELISA/dot blot:  Antibody dilution 1:20,000-1:50,000. Western blot:  Antibody dilution 1:500-1,000 in DiluObuffer.  IMM:  n.d; IHC n.d

Reactivity                The antibodies Mad (Mad-101AP) labels 56kDa Mothers Against decapentaplegic (dpp) protein (MAD) in PC-Mad samples.

Protocols:                Description and use of this antibody in various applications is provided with the product.  Standard protocol for various applications (Western blot; immunoprecipitation and immunohistochemistry) of this antibody can be requested by calling our Technical support line.  The recommended dilutions are for reference only and FabGennix Inc. strongly recommends investigators to optimize conditions for use of this product in their laboratories.  

Form/Storage:       The antiserum is supplied in antibody stabilization buffer with preservatives.  For long-term storage of antibody, store at -20^oC FabGennix Inc. does not recommend storage of very dilute antibody solutions unless they are prepared in specially formulated multi use antibody dilution buffer (Cat # DiluOBuffer).  Working solutions of antibodies in DiluOBuffer should be filtered through 0.45um filter after every use for long-term storage.

 

 

For research use only, not for diagnostic or therapeutic use.