Monoclonal Antibodies: Mad1, Mad2, HIRA

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Murine Monoclonal Antibodies to Mad1, and Mad2—Mitotic Checkpoint Proteins

The spindle checkpoint complex is composed of Mad1, Mad2 and Mad3.

Mad1 negatively regulates mitosis by preventing cells with misaligned chromosomes from dividing. It is an important target in cancer research because it may sensitize cells to existing chemotherapeutics and its expression may be an indicator of cellular sensitivity to chemotherapy. Mad 1is known to repress Myc target genes and antagonize Myc function .

Mad2 provides feedback control that prevents cells with incompletely assembled spindles from leaving mitosis. Researchers have found that deletion of a single Mad2 allele results in a defective mitotic checkpoint; Mad2+/- mice develop lung tumors at high rates after long latencies, implicating defects in the mitotic checkpoint of tumorigenesis . Mad2 is an important target in cancer research because it may sensitize cells to existing chemotherapeutics and its expression may be an indicator of cellular sensitivity to chemotherapy.

Mad1

ApplicationsIF and IP. Not effective for WB.
  
Specificity:Reacts with human Mad1; other species specificity has not yet been determined
  
Immunogen:Recombinant human Mad1
  
Isotype:IgG2b
  
Citation:*Weaver, BA, et al. "Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss," (2003) Journal of Cell Biology, 162: 551-563. [PMID: 12925705]
*Liu ST, Rattner JB, et al. "Mapping the assembly pathways that specify formation of the trilaminar kinetochore plates in human cells," (2006) Journal of Cell Biology, 175(1):41-53 [PMID: 17030981]
*Huang H, Feng J, et al. "Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments," (2007) Journal of Cell Biology 7;177(3):413-24 [PMID: 17485487]

Mad2

ApplicationsWB and IP. Not effective for IF
  
Specificity:Western blot with a variety of human cell lines confirms that the antibody is specific for human Mad2; other species specificity has not yet been determined
  
Immunogen:Recombinant human Mad2
  
Isotype:IgG1
  
Citation:Braunstein I, Miniowitz S, et al., "Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint," Proc Natl Acad Sci USA, 104(12):4870-5 [PMID: 17360335]

Monoclonal Antibodies against human HIRA, a novel cell cycle regulator

HIRA is the human homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. In yeast, Hir1p and Hir2p control histone gene expression through the cell cycle. In human cells, HIRA contributes to formation of transcriptionally-repressive heterochromatin in senescent cells and also has roles in transcription-coupled chromatin assembly.

Dr. Peter Adams and colleagues of Fox Chase Cancer Center have developed murine monoclonal antibodies against human HIRA.

ClonesWC15, WC19, WC117, WC119
  
Applications:Clone WC15 works best for IP
Clone WC119 works best for WB
A cocktail of all four mAbs works well in IF
  
Specificity:Each reacts with human HIRA. WC15 and WC119 also react with mouse and X. laevis proteins (Tagami H et al. 2004. Cell 116: 51-61 [PMID:14718166]). Reactivity against other species has not yet been fully characterized
  
Immunogen:Glutathione S-transferase-HIRA [421-729] (human HIRA)
  
Isotype:

*Hall C, Nelson DM, et al. "HIRA, the human homologue of yeast Hir1p and Hir2p, is a novel cyclin-cdk2 substrate whose expression blocks S-phase progression," Mol. Cell. Biol. 2001 Mar;21(5):1854-65 [PMID: 11238922

*Zhang R, Poustovoitov MV, et al. "Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA," Dev Cell. 2005 Jan;8(1):19-30 [PMID: 15621527]