The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
1/100 - 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
1/2000 - 1/10000. Detects a band of approximately 120 kDa (predicted molecular weight: 120 kDa).
Use at 2-5 µg/mg of lysate.
Essential component of the mitotic checkpoint. Required for normal mitosis progression. The mitotic checkpoint delays anaphase until all chromosomes are properly attached to the mitotic spindle. One of its checkpoint functions is to inhibit the activity of the anaphase-promoting complex/cyclosome (APC/C) by blocking the binding of CDC20 to APC/C, independently of its kinase activity. The other is to monitor kinetochore activities that depend on the kinetochore motor CENPE. Required for kinetochore localization of CENPE. Negatively regulates PLK1 activity in interphase cells and suppresses centrosome amplification. Also implicated in triggering apoptosis in polyploid cells that exit aberrantly from mitotic arrest. May play a role for tumor suppression.
Highly expressed in thymus followed by spleen. Preferentially expressed in tissues with a high mitotic index.
Involvement in disease
Note=Defects in BUB1B are associated with tumor formation. Defects in BUB1B are the cause of premature chromatid separation trait (PCS) [MIM:176430]. PCS consists of separate and splayed chromatids with discernible centromeres and involves all or most chromosomes of a metaphase. It is found in up to 2% of metaphases in cultured lymphocytes from approximately 40% of normal individuals. When PCS is present in 5% or more of cells, it is known as the heterozygous PCS trait and has no obvious phenotypic effect, although some have reported decreased fertility. Inheritance is autosomal dominant. Defects in BUB1B are the cause of mosaic variegated aneuploidy syndrome (MVA) [MIM:257300]. MVA is a severe autosomal recessive developmental disorder characterized by mosaic aneuploidies, predominantly trisomies and monosomies, involving multiple different chromosomes and tissues. The proportion of aneuploid cells varies but is usually more than 25% and is substantially greater than in normal individuals. Affected individuals typically present with severe intrauterine growth retardation and microcephaly. Eye anomalies, mild dysmorphism, variable developmental delay, and a broad spectrum of additional congenital abnormalities and medical conditions may also occur. The risk of malignancy is high, with rhabdomyosarcoma, Wilms tumor and leukemia reported in several cases. MVA is caused by biallelic mutations in the BUB1B gene.
Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. BUB1 subfamily. Contains 1 BUB1 N-terminal domain. Contains 1 protein kinase domain.
The D-box targets the protein for rapid degradation by ubiquitin-dependent proteolysis during the transition from mitosis to interphase. The BUB1 N-terminal domain directs kinetochore localization and binding to BUB3.
Proteolytically cleaved by caspase-3 in a cell cycle specific manner. The cleavage might be involved in the durability of the cell cycle delay. Caspase-3 cleavage is associated with abrogation of the mitotic checkpoint. The major site of cleavage is at Asp-610. Acetylation at Lys-250 regulates its degradation and timing in anaphase entry. Ubiquitinated. Degradated by the proteasome. Sumoylated by SUMO2 and SUMO3. The sumoylation mediates the association with CENPE at the kinetochore. Autophosphorylated in vitro. Intramolecular autophosphorylation is stimulated by CENPE. Phosphorylated during mitosis and hyperphosphorylated in mitotically arrested cells. Phosphorylation at Ser-670 and Ser-1043 occurs at kinetochores upon mitotic entry with dephosphorylation at the onset of anaphase.
Cytoplasm. Nucleus. Chromosome > centromere > kinetochore. Cytoplasm > cytoskeleton > centrosome. Cytoplasmic in interphase cells. Associates with the kinetochores in early prophase. Kinetochore localization requires BUB1, PLK1 and CASC5.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labelling BubR1 with ab70544 at 1/200 (1µg/ml). Detection: DAB.
Western blot - BubR1 antibody (ab70544)
All lanes : Anti-BubR1 antibody (ab70544) at 0.04 µg/ml
Lane 1 : Whole cell lysate from HeLa cells at 50 µg Lane 2 : Whole cell lysate from HeLa cells at 15 µg Lane 3 : Whole cell lysate from HeLa cells at 5 µg Lane 4 : Whole cell lysate from 293T cells at 50 µg
Developed using the ECL technique.
Predicted band size: 120 kDa Observed band size: 120 kDa
Exposure time: 30 seconds
Immunoprecipitation - BubR1 antibody (ab70544)
1mg whole cell lysate from HeLa cells was immunoprecipitated with ab70544 at 3ug/mg of lysate (lane 1) or a with a control rabbit IgG (lane 2). For the subsequent western blot, 20% of the immunoprecipitae was loaded per lane, and ab70544 was used at 1ug/ml. Detection: chemiluminescence with exposure time of 3 seconds.