Recombinant Anti-Bub1 antibody [EPR18947] (ab195268)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR18947] to Bub1
- Suitable for: WB, IHC-P, ICC/IF
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-Bub1 antibody [EPR18947]
See all Bub1 primary antibodies -
Description
Rabbit monoclonal [EPR18947] to Bub1 -
Host species
Rabbit -
Specificity
ab195268 shows stronger signal in mouse and rat testis tissues but weaker in the human testis. -
Tested applications
Suitable for: WB, IHC-P, ICC/IFmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HeLa, K562, U-2 OS, F9 and mESC whole cell lysates; Human testis and fetal liver lysates; Mouse and Rat testis lysates. IHC-P: Mouse and Rat testis tissues.
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General notes
Our in house IHC testing showed positive staining in testis tissue ONLY. Other tissues were negative.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR18947 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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KO cell lines
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KO cell lysates
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab195268 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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WB |
1/1000. Detects a band of approximately 122 kDa (predicted molecular weight: 122 kDa).
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IHC-P |
1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Our in house IHC testing showed positive staining in testis tissue ONLY. Other tissues were negative. |
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ICC/IF |
1/100.
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Notes |
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WB
1/1000. Detects a band of approximately 122 kDa (predicted molecular weight: 122 kDa). |
IHC-P
1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. Our in house IHC testing showed positive staining in testis tissue ONLY. Other tissues were negative. |
ICC/IF
1/100. |
Target
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Function
Serine/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 specificity
High 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 similarities
Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. BUB1 subfamily.
Contains 1 BUB1 N-terminal domain.
Contains 1 protein kinase domain. -
Domain
The 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 localization
Nucleus. 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. - Information by UniProt
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Database links
- Entrez Gene: 699 Human
- Entrez Gene: 12235 Mouse
- Entrez Gene: 296137 Rat
- Omim: 602452 Human
- SwissProt: O43683 Human
- SwissProt: O08901 Mouse
- Unigene: 469649 Human
- Unigene: 2185 Mouse
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Alternative names
- Bub1 antibody
- BUB1 budding uninhibited by benzimidazoles 1 homolog antibody
- BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) antibody
see all
Images
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All lanes : Anti-Bub1 antibody [EPR18947] (ab195268) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : BUB1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 122 kDa
Observed band size: 125 kDa why is the actual band size different from the predicted?Lanes 1- 2: Merged signal (red and green). Green - ab195268 observed at 125 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab195268 was shown to react with Bub1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265145 (knockout cell lysate ab257373) was used. Wild-type HeLa and BUB1 knockout HeLa cell lysates were subjected to SDS-PAGE. ab195268 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Bub1 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: F9 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab195268 observed at 125 kDa. Red - loading control, ab8245, observed at 37 kDa.ab195268 was shown to recognize Bub1 when Bub1 knockout samples were used, along with additional cross-reactive bands. Wild-type and Bub1 knockout samples were subjected to SDS-PAGE. Ab195268 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10,000 dilution respectively and incubated overnight at 4C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ( ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.
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Ab195268 staining Bub1 in HeLa (human cervix adenocarcinoma epithelial cell) cells by Immunocytochemistry. Cells were fixed with 4% paraformaldehyde and permeabilised with 0.1% TritonX-100. Samples were incubated with primary antibody at 1:100 dilution (7.8 μg/ml). Alexa Fluor® 594 Anti-alpha tubulin [DM1A] – Microtubule Marker (ab195889) was used as the counterstain antibody at 1:200 dilution (2.5 μg/ml). An AlexaFluor®488 Goat anti-Rabbit (ab150077) was used as a secondary antibody at 1:1000 dilution (2µg/ml). DAPI was used as a nuclear counterstain. Confocal image showing positive kinetochore staining in HeLa cells in M phase.
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Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling Bub1 with ab195268 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Nucleus staining on Rat testis is observed.
Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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All lanes : Anti-Bub1 antibody [EPR18947] (ab195268) at 1/1000 dilution
Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 3 : U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 122 kDa
Observed band size: 122 kDaBlocking/Dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: Lane 1 & 2: 10 seconds; Lane 2: 3 minutes.
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All lanes : Anti-Bub1 antibody [EPR18947] (ab195268) at 1/1000 dilution
Lane 1 : Human testis lysate
Lane 2 : Human fetal liver lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution
Predicted band size: 122 kDa
Observed band size: 122 kDa
Exposure time: 3 minutesBlocking/Dilution buffer: 5% NFDM/TBST.
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All lanes : Anti-Bub1 antibody [EPR18947] (ab195268) at 1/1000 dilution
Lane 1 : Mouse testis lysate
Lane 2 : Rat testis lysate
Lane 3 : F9 (Mouse embryonic testicular cancer cell line) whole cell lysate
Lane 4 : mESC (Mouse embryonic stem cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 122 kDa
Observed band size: 122 kDa
Exposure time: 3 minutesBlocking/Dilution buffer: 5% NFDM/TBST.
The molecular weight observed is consistent with what has been described in the literature PMID:17189386
PMID:16864798 -
Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling Bub1 with ab195268 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Nucleus staining on Mouse testis is observed.
Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (8)
ab195268 has been referenced in 8 publications.
- Lou L et al. Identification of hub genes and construction of prognostic nomogram for patients with Wilms tumors. Front Oncol 12:982110 (2022). PubMed: 36338682
- Edwards DM et al. Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia. Front Oncol 11:752933 (2021). PubMed: 34804941
- Song X et al. Counteraction between Astrin-PP1 and Cyclin-B-CDK1 pathways protects chromosome-microtubule attachments independent of biorientation. Nat Commun 12:7010 (2021). PubMed: 34853300
- Zhu LJ et al. BUB1 promotes proliferation of liver cancer cells by activating SMAD2 phosphorylation. Oncol Lett 19:3506-3512 (2020). PubMed: 32269624
- Zhou C et al. The cohesin release factor Wapl interacts with Bub3 to govern SAC activity in female meiosis I. Sci Adv 6:eaax3969 (2020). PubMed: 32284991
- Lu Y et al. Characteristic Analysis of Featured Genes Associated With Stemness Indices in Colorectal Cancer. Front Mol Biosci 7:563922 (2020). PubMed: 33134313
- Wu J et al. Overexpression of hsa-miR-186 induces chromosomal instability in arsenic-exposed human keratinocytes. Toxicol Appl Pharmacol 378:114614 (2019). PubMed: 31176655
- Stahl D et al. Low BUB1 expression is an adverse prognostic marker in gastric adenocarcinoma. Oncotarget 8:76329-76339 (2017). IHC-P ; Human . PubMed: 29100315