概述

  • 产品名称Anti-BubR1抗体[8G1]
    参阅全部 BubR1 一抗
  • 描述
    小鼠单克隆抗体[8G1] to BubR1
  • 经测试应用适用于: IP, ICC/IF, WBmore details
  • 种属反应性
    与反应: Human
  • 免疫原

    N-terminal tagged fusion recombinant fragment, corresponding to amino acids 1-350 of Human BubR1.

  • 阳性对照
    • Recent batches of this antibody have given a positive signal in WB against recombinant BubR1 however we have been unable to detect endogenous BubR1 in lysates tested. Previous batches of this antibody have given a positive signal in HeLa nuclear lysate (see tested notes section for protocol details). For further information please contact our Scientific Support Team

性能

应用

Our Abpromise guarantee covers the use of ab4637 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用 Ab评论 说明
IP Use at an assay dependent dilution.
ICC/IF 1/200.
WB Use a concentration of 10 µg/ml. Detects a band of approximately 130 kDa (predicted molecular weight: 120 kDa). Abcam recommends using milk as the blocking agent. We would suggest using the following protocol; arrest HeLa cells in mitosis with 60ng/ml of sterile nocodazole. Harvest cells by mechanical shake-off between 12 to 16 hours after drug addition. Pellet cells, wash in PBS, and lyse in RIPA. Centrifuge lysates at 4C/15,000g for 10 minutes. Store supernatants and determine protein concentration. Load 40 to 50ug of total protein per lane.

靶标

  • 功能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.
  • 组织特异性Highly expressed in thymus followed by spleen. Preferentially expressed in tissues with a high mitotic index.
  • 疾病相关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.
  • 序列相似性Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. BUB1 subfamily.
    Contains 1 BUB1 N-terminal domain.
    Contains 1 protein kinase domain.
  • 结构域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.
  • 翻译后修饰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.
  • 细胞定位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.
  • Information by UniProt
  • 数据库链接
  • 别名
    • Beta homolg of S. cerevisiae BUB 1 antibody
    • Beta homolg of S. cerevisiae budding uninhibited by benzimidazoles antibody
    • BUB 1B antibody
    • BUB1 budding uninhibited by benzimidazoles 1 homolog beta antibody
    • Bub1A antibody
    • BUB1B antibody
    • BUB1B_HUMAN antibody
    • BUB1beta antibody
    • BUBR1 antibody
    • Budding Uninhibited by Benzimidazoles 1 beta antibody
    • Budding uninhibited by benzimidazoles 1 homolog beta (yeast) antibody
    • hBUBR1 antibody
    • MAD3/BUB1 related protein kinase antibody
    • MAD3/BUB1-related protein kinase antibody
    • MAD3L antibody
    • Mitotic checkpoint gene BUB1B antibody
    • Mitotic checkpoint kinase MAD3L antibody
    • Mitotic checkpoint serine/threonine protein kinase BUB1 beta antibody
    • Mitotic checkpoint serine/threonine-protein kinase BUB1 beta antibody
    • MVA1 antibody
    • OTTHUMP00000160319 antibody
    • Protein SSK1 antibody
    • SSK 1 antibody
    • SSK1 antibody
    see all

Anti-BubR1 antibody [8G1] 图像

  • Immunofluorescent imaging of an asynchronous cycling population of human cells (U2OS) with ab4637 strikingly confirms the specificity of this antibody.  No signal is detected from interphase cells, whereas cells undergoing mitosis accumulate BubR1 at the kinetochores.  Image reveals kinetochores at prometaphase, and exactly agrees with numerous published images of BubR1 (see for example Johnson et al J Cell Sci. 117(Pt 8):1577-89 (2004). 

    IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour.  Primary
    antibody used at 1/100 in 5% milk / 0.2% TWEEN for one hour, secondary antibody for 30 minutes.  All blocking and incubation steps carried out at 37 degrees C.

  • HeLa cells were stained with anti-BubR1 (ab4637) in green and DAPI in blue in panel one and anti-BubR1 (green) and SH-CREST (red) to stain the centromeres in panel 2. Fix cells for 30 minutes on ice in 4% formaldehyde in PEM. Quench autofluorescence 2 x 5 min. with 1 mg/ml Na borohydride OR 100 mM ammonium chloride in PEM. Permeablize 30 min. with 0.5% TX-100 in PEM. Block 30 minutes in 5% milk in TBST. Primary antibody was incubated 1/200 overnight at 4oC diluted in 5% milk in TBST. Secondary antibody was incubated for 1 hour at RT diluted in 5% milk in TBST. Post-fix 20 min. on ice in 4% formaldehyde in PEM. Quench autofluorescence 2 x 5 min. with ammonium chloride in PEM. Counterstain with DAPI in TBST. Mount with ProLong Gold antifade reagent from Invitrogen. Notes: Ample washing between each step. TBST = Tris buffered saline + 0.1% Tween. PEM = 80 mM K-PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl2.
  • Immunofluorescence of nocodazole treated HeLa cells with ab4637 staining BubR1 (green). CREST was used to mark centromeres (red) and the DNA is stained with DAPI (blue).

    Cells were fixed with PFA 4% and permealized with Triton X-100 0.1%. Blocking was done with 4% BSA. ab4637 was used at a 1:200 dilution in 4% BSA for 1.5h at room temperature. Secondary antibodies were incubated for 45min. Coverslips were then stained with DAPI and mounted with Mowiol mounting media.

    ab4637 reveals BubR1 in mitotic cells colocalizing with kinetochores, as expected.



  • Predicted band size : 120 kDa

Anti-BubR1 antibody [8G1] (ab4637)参考文献

This product has been referenced in:
  • Mäki-Jouppila JH  et al. Centmitor-1, a novel acridinyl-acetohydrazide, possesses similar molecular interaction field and antimitotic cellular phenotype as rigosertib, on 01910.Na. Mol Cancer Ther 13:1054-66 (2014). ICC/IF ; Human . Read more (PubMed: 24748653) »
  • Salmela AL  et al. Novel pyrimidine-2,4-diamine derivative suppresses the cell viability and spindle assembly checkpoint activity by targeting Aurora kinases. Carcinogenesis 34:436-45 (2013). Read more (PubMed: 23104179) »

See all 15 Publications for this product

Product Wall

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 22°C
Sample Human Cell (293T)
Specification 293T
Permeabilization Yes - 0.5% Triton X100
Fixative Paraformaldehyde
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Abcam user community

Verified customer

提交于 Jan 21 2015

Thank you for your note.  This is to let you know that I have just contacted and asked our Account Department to raise a credit note for you - for the cost of one vial of ab4637. For your information, the internal reference note for this credit is CNXX...

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Thank you for your enquiry regarding ab4637 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody. Currently, we are retesting this antibody and i...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa)
Specification HeLa
Fixative 20mM PIPES pH 6.8, 4% formaldehyde, 0.2% Triton-X 100, 10mM EGTA, 1mM MgCl2
Permeabilization No
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 20°C
Username

Dr. Kum Kum Khanna

Verified customer

提交于 Jun 18 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa)
Specification HeLa
Fixative Methanol
Permeabilization No
Username

Dr. Kirk McManus

Verified customer

提交于 Jun 13 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa)
Specification HeLa
Fixative Methanol
Permeabilization No
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

提交于 Apr 11 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (Hela)
Specification Hela
Fixative Paraformaldehyde
Permeabilization Yes - 0.5% CHAPS
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 20% · Temperature: 23°C
Username

Abcam user community

Verified customer

提交于 Feb 01 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (breast cancer)
Specification breast cancer
Fixative Paraformaldehyde
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
Username

Dr. Lee Bergman

Verified customer

提交于 Jun 28 2006

Thank you for your email. The replacement vial of ab4637 is being sent to your attention on order# 75703 and you should receive this tomorrow. Please let me know how it works out for you and if you have any additional questions.

Thank you for the details that you have provided. Looking through our records for this antibody, the first batch that you received is the same as this latest one. My feeling is that this most recent vial went off during shipping. I can certainly send y...

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