The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/5000 - 1/15000. Detects a band of approximately 140 kDa (predicted molecular weight: 122 kDa).
1/500 - 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Use at 1-4 µg/mg of lysate.
FunctionSerine/threonine-protein kinase that performs 2 crucial functions during mitosis: it is essential for spindle-assembly checkpoint signaling and for correct chromosome alignment. Has a key role in the assembly of checkpoint proteins at the kinetochore, being required for the subsequent localization of CENPF, BUB1B, CENPE and MAD2L1. Required for the kinetochore localization of PLK1. Plays an important role in defining SGOL1 localization and thereby affects sister chromatid cohesion. Acts as a substrate for anaphase-promoting complex or cyclosome (APC/C) in complex with its activator CDH1 (APC/C-Cdh1). Necessary for ensuring proper chromosome segregation and binding to BUB3 is essential for this function. Can regulate chromosome segregation in a kinetochore-independent manner. Can phosphorylate BUB3. The BUB1-BUB3 complex plays a role in the inhibition of APC/C when spindle-assembly checkpoint is activated and inhibits the ubiquitin ligase activity of APC/C by phosphorylating its activator CDC20. This complex can also phosphorylate MAD1L1. Kinase activity is essential for inhibition of APC/CCDC20 and for chromosome alignment but does not play a major role in the spindle-assembly checkpoint activity. Mediates cell death in response to chromosome missegregation and acts to suppress spontaneous tumorigenesis.
Tissue specificityHigh expression in testis and thymus, less in colon, spleen, lung and small intestine. Expressed in fetal thymus, bone marrow, heart, liver, spleen and thymus. Expression is associated with cells/tissues with a high mitotic index.
Sequence similaritiesBelongs to the protein kinase superfamily. Ser/Thr protein kinase family. BUB1 subfamily. Contains 1 BUB1 N-terminal domain. Contains 1 protein kinase domain.
DomainThe KEN box is required for its ubiquitination and degradation. BUB1 N-terminal domain directs kinetochore localization and binding to BUB3.
Post-translational modificationsPhosphorylated upon DNA damage, probably by ATM or ATR. Upon spindle-assembly checkpoint activation it is hyperphosphorylated and its kinase activity toward CDC20 is stimulated. Phosphorylation at Thr-609 is required for interaction with PLK1, phosphorylation at this site probably creates a binding site for the POLO-box domain of PLK1, thus enhancing the PLK1-BUB1 interaction. Ubiquitinated and degraded during mitotic exit by APC/C-Cdh1.
Cellular localizationNucleus. Chromosome > centromere > kinetochore. Nuclear in interphase cells. Accumulates gradually during G1 and S phase of the cell cycle, peaks at G2/M, and drops dramatically after mitosis. Localizes to the outer kinetochore. Kinetochore localization is required for normal mitotic timing and checkpoint response to spindle damage and occurs very early in prophase. AURKB, CASC5 and INCENP are required for kinetochore localization.
Detection of Human Bub1 by Immunoprecipitiaton. HeLa Whole cell lysate used at 50 µg for WB Input; 1 mg for IP. Cells were asynchronously growing (-) or treated with Nocodozale (Noc +). ab70372 was used at 1 µg/mg lysate for IP and at 0.1 µg/ml for subsequent Western blot detection .
References for Anti-Bub1 antibody (ab70372)
This product has been referenced in:
Sun Z et al. Kaposi's sarcoma-associated herpesvirus-encoded LANA can induce chromosomal instability through targeted degradation of the mitotic checkpoint kinase Bub1. J Virol88:7367-78 (2014).
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