Recombinant Anti-Rb antibody [E182] (ab32513)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E182] to Rb
- Suitable for: Flow Cyt (Intra), ICC/IF, IP, WB
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-Rb antibody [E182]
See all Rb primary antibodies -
Description
Rabbit monoclonal [E182] to Rb -
Host species
Rabbit -
Specificity
The antibody detects pan Rb protein. -
Tested applications
Suitable for: Flow Cyt (Intra), ICC/IF, IP, WBmore details
Unsuitable for: IHC -
Species reactivity
Reacts with: Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Hek293, and Jurkat cell lysates; ICC/IF: A431 cells; Flow Cyt (intra): Jurkat cells.
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General notes
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.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
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: 7.20
Preservative: 0.01% Sodium azide
Constituents: 40% Glycerol (glycerin, glycerine), 0.05% BSA, 59% PBS -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
E182 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Isotype control
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Positive Controls
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32513 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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Flow Cyt (Intra) |
1/70.
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ICC/IF |
1/50.
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IP |
1/40.
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WB |
1/1000. Detects a band of approximately 110 kDa (predicted molecular weight: 106 kDa).
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Notes |
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Flow Cyt (Intra)
1/70. |
ICC/IF
1/50. |
IP
1/40. |
WB
1/1000. Detects a band of approximately 110 kDa (predicted molecular weight: 106 kDa). |
Target
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Function
Key regulator of entry into cell division that acts as a tumor suppressor. Promotes G0-G1 transition when phosphorylated by CDK3/cyclin-C. Acts as a transcription repressor of E2F1 target genes. The underphosphorylated, active form of RB1 interacts with E2F1 and represses its transcription activity, leading to cell cycle arrest. Directly involved in heterochromatin formation by maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin by stabilizing histone methylation. Recruits and targets histone methyltransferases SUV39H1, KMT5B and KMT5C, leading to epigenetic transcriptional repression. Controls histone H4 'Lys-20' trimethylation. Inhibits the intrinsic kinase activity of TAF1. Mediates transcriptional repression by SMARCA4/BRG1 by recruiting a histone deacetylase (HDAC) complex to the c-FOS promoter. In resting neurons, transcription of the c-FOS promoter is inhibited by BRG1-dependent recruitment of a phospho-RB1-HDAC1 repressor complex. Upon calcium influx, RB1 is dephosphorylated by calcineurin, which leads to release of the repressor complex (By similarity). In case of viral infections, interactions with SV40 large T antigen, HPV E7 protein or adenovirus E1A protein induce the disassembly of RB1-E2F1 complex thereby disrupting RB1's activity. -
Tissue specificity
Expressed in the retina. -
Involvement in disease
Childhood cancer retinoblastoma
Bladder cancer
Osteogenic sarcoma -
Sequence similarities
Belongs to the retinoblastoma protein (RB) family. -
Domain
The Pocket domain binds to the threonine-phosphorylated domain C, thereby preventing interaction with heterodimeric E2F/DP transcription factor complexes. -
Post-translational
modificationsPhosphorylated by CDK6 and CDK4, and subsequently by CDK2 at Ser-567 in G1, thereby releasing E2F1 which is then able to activate cell growth. Dephosphorylated at the late M phase. SV40 large T antigen, HPV E7 and adenovirus E1A bind to the underphosphorylated, active form of pRb. Phosphorylation at Thr-821 and Thr-826 promotes interaction between the C-terminal domain C and the Pocket domain, and thereby inhibits interactions with heterodimeric E2F/DP transcription factor complexes. Dephosphorylated at Ser-795 by calcineruin upon calcium stimulation. CDK3/cyclin-C-mediated phosphorylation at Ser-807 and Ser-811 is required for G0-G1 transition. Phosphorylated by CDK1 and CDK2 upon TGFB1-mediated apoptosis.
N-terminus is methylated by METTL11A/NTM1 (By similarity). Monomethylation at Lys-810 by SMYD2 enhances phosphorylation at Ser-807 and Ser-811, and promotes cell cycle progression. Monomethylation at Lys-860 by SMYD2 promotes interaction with L3MBTL1.
Acetylation at Lys-873 and Lys-874 regulates subcellular localization, at least during keratinocytes differentiation. -
Cellular localization
Nucleus. - Information by UniProt
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Database links
- Entrez Gene: 5925 Human
- Omim: 614041 Human
- SwissProt: P06400 Human
- Unigene: 408528 Human
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Alternative names
- Exon 17 tumor GOS561 substitution mutation causes premature stop antibody
- GOS563 exon 17 substitution mutation causes premature stop antibody
- OSRC antibody
see all
Images
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Purified ab32513 at 1/40 dilution (2µg) immunoprecipitating Rb in Jurkat whole cell lysate.
Lane 1 (input): Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10µg
Lane 2 (+): ab32513 + Jurkat whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32513 in Jurkat whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 120 kDa -
All lanes : Anti-Rb antibody [E182] (ab32513) at 1/1000 dilution (Purified)
Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) prepared in RIPA lysis method whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia T lymphocyte) prepared in 1% SDS Hot lysis method whole cell lysate
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 106 kDa
Observed band size: 106-120 kDa why is the actual band size different from the predicted?1% SDS Hot lysis method is preferred for this antibody.
Blocking Buffer and concentration: 5% NFDM/TBST
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Immunocytochemistry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling Rb with Purified ab32513 at 1:50 dilution (10 ?g/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
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Intracellular Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling Rb with Purified ab32513 at 1/70 dilution (10µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).
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Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: Rb knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: HEK293 whole cell lysate (20 µg)Lanes 1 - 4: Merged signal (red and green). Green - ab32513 observed at 120 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32513 was shown to recognize Rb when Rb knockout samples were used, along with additional cross-reactive bands. Wild-type and Rb knockout samples were subjected to SDS-PAGE. Ab32513 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (3)
ab32513 has been referenced in 3 publications.
- Kumari R et al. Simultaneous expression of MMB-FOXM1 complex components enables efficient bypass of senescence. Sci Rep 11:21506 (2021). PubMed: 34728711
- Richardson E et al. Mechanism-based screen establishes signalling framework for DNA damage-associated G1 checkpoint response. PLoS One 7:e31627 (2012). ICC/IF ; Human . PubMed: 22384045
- Haller F et al. Increased KIT signalling with up-regulation of cyclin D correlates to accelerated proliferation and shorter disease-free survival in gastrointestinal stromal tumours (GISTs) with KIT exon 11 deletions. J Pathol 216:225-35 (2008). PubMed: 18729075