The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml.
1/1000 - 1/5000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.
1/1000. Detects a band of approximately 24 kDa (predicted molecular weight: 24 kDa).
Use at 2-4 µg/mg of lysate.
FunctionComponent of the spindle-assembly checkpoint that prevents the onset of anaphase until all chromosomes are properly aligned at the metaphase plate. Required for the execution of the mitotic checkpoint which monitors the process of kinetochore-spindle attachment and inhibits the activity of the anaphase promoting complex by sequestering CDC20 until all chromosomes are aligned at the metaphase plate.
Sequence similaritiesBelongs to the MAD2 family. Contains 1 HORMA domain.
DomainThe protein has two highly different native conformations, an inactive open conformation that cannot bind CDC20 and that predominates in cytosolic monomers, and an active closed conformation. The protein in the closed conformation preferentially dimerizes with another molecule in the open conformation, but can also form a dimer with a molecule in the closed conformation. Formation of a heterotetrameric core complex containing two molecules of MAD1L1 and of MAD2L1 in the closed conformation promotes binding of another molecule of MAD2L1 in the open conformation and the conversion of the open to the closed form, and thereby promotes interaction with CDC20.
Post-translational modificationsPhosphorylated on multiple serine residues. The level of phosphorylation varies during the cell cycle and is highest during mitosis. Phosphorylation abolishes interaction with MAD1L1 and reduces interaction with CDC20. Phosphorylated by NEK2.
Cellular localizationNucleus. Chromosome > centromere > kinetochore. Cytoplasm. Cytoplasm > cytoskeleton > spindle pole. Recruited by MAD1L1 to unattached kinetochores (Probable). Recruited to the nuclear pore complex by TPR during interphase. Recruited to kinetochores in late prometaphase after BUB1, CENPF, BUB1B and CENPE. Kinetochore association requires the presence of NEK2. Kinetochore association is repressed by UBD.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue labelling MAD2 with ab70383 at 1/1000 (1µg/ml). Detection: DAB.
Western blot - MAD2 antibody (ab70383)
Anti-MAD2 antibody (ab70383) at 1 µg/ml + NETN whole cell lysate from HeLa cells at 50 µg Developed using the ECL technique
Predicted band size : 24 kDa Observed band size : 24 kDa Additional bands at : 38 kDa. We are unsure as to the identity of these extra bands.
Exposure time : 1 minute
Immunoprecipitation - MAD2 antibody (ab70383)This image is courtesy of an anonymous Abreview
ab70383 Immunoprecipitating MAD2 in human HeLa whole cell lysate. 200µg of cell lysate was incubated with primary antibody (2 µg/ml in NETN buffer) and matrix (Protein A/G) for 16 hours at 4°C. For western blotting ab70383 (1/1000) was used to confirm successful immunoprecipation.
ICC/IF image of ab70383 stained MCF7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab70383, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
References for Anti-MAD2 antibody (ab70383)
This product has been referenced in:
Eliezer Y et al. Interplay between the DNA damage proteins MDC1 and ATM in the regulation of the spindle assembly checkpoint. J Biol Chem289:8182-93 (2014).
Read more (PubMed: 24509855) »
Rajanala K et al. Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function. J Cell Sci127:3505-20 (2014).
Read more (PubMed: 24938596) »