Recombinant Anti-Bmi1 antibody [EPR3745(2)] - BSA and Azide free (ab216444)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR3745(2)] to Bmi1 - BSA and Azide free
- Suitable for: IP, IHC-P, WB, ICC/IF, ChIC/CUT&RUN-seq
- Knockout validated
- Reacts with: Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-Bmi1 antibody [EPR3745(2)] - BSA and Azide free
See all Bmi1 primary antibodies -
Description
Rabbit monoclonal [EPR3745(2)] to Bmi1 - BSA and Azide free -
Host species
Rabbit -
Tested applications
Suitable for: IP, IHC-P, WB, ICC/IF, ChIC/CUT&RUN-seqmore details -
Species reactivity
Reacts with: Rat, Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: MCF7, A431, HEK293T, K562, SAOS-2, SW480, MOLT4, PC-12 and HT1080 cell lysates. IHC-P: Human tonsil, colonic adenocarcinoma, lung adenocarcinoma, breast carcinoma and thyroid gland carcinoma tissues. ICC/IF: SW480 and HeLa cells. IP: K-562 cell lysate ChIC/CUT&RUN-Seq: NCCIT cells.
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General notes
ab216444 is the carrier-free version of ab126783.
Mouse: Internal data indicated that the antibody is not suitable for WB application in mouse species.
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.
This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.
Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.
This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.
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. Do Not Freeze. -
Storage buffer
pH: 7.20
Constituent: PBS -
Carrier free
Yes -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR3745(2) -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Conjugation kits
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Isotype control
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KO cell lines
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KO cell lysates
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Positive Controls
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab216444 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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IP |
Use at an assay dependent concentration.
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IHC-P |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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WB |
Use at an assay dependent concentration. Detects a band of approximately 40 kDa (predicted molecular weight: 36 kDa).
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ICC/IF |
Use at an assay dependent concentration.
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ChIC/CUT&RUN-seq |
Use at an assay dependent concentration.
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Notes |
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IP
Use at an assay dependent concentration. |
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. |
WB
Use at an assay dependent concentration. Detects a band of approximately 40 kDa (predicted molecular weight: 36 kDa). |
ICC/IF
Use at an assay dependent concentration. |
ChIC/CUT&RUN-seq
Use at an assay dependent concentration. |
Target
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Function
Component of the Polycomb group (PcG) multiprotein PRC1 complex, a complex required to maintain the transcriptionally repressive state of many genes, including Hox genes, throughout development. PcG PRC1 complex acts via chromatin remodeling and modification of histones; it mediates monoubiquitination of histone H2A 'Lys-119', rendering chromatin heritably changed in its expressibility. In the PRC1 complex, it is required to stimulate the E3 ubiquitin-protein ligase activity of RNF2/RING2. -
Sequence similarities
Contains 1 RING-type zinc finger. -
Post-translational
modificationsMonoubiquitinated (By similarity). May be polyubiquitinated; which does not lead to proteasomal degradation. -
Cellular localization
Nucleus. Cytoplasm. - Information by UniProt
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Database links
- Entrez Gene: 100532731 Human
- Entrez Gene: 648 Human
- Entrez Gene: 307151 Rat
- Omim: 164831 Human
- SwissProt: P35226 Human
- Unigene: 380403 Human
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Alternative names
- B lymphoma Mo MLV insertion region (mouse) antibody
- B lymphoma Mo MLV insertion region 1 homolog antibody
- Bmi 1 antibody
see all
Images
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ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 NCCIT (Human pluripotent embryonic carcinoma cell line) cells and 5 µg of ab126783 [EPR3745(2)]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.
Additional screenshots of mapped reads can be downloaded here.
The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.
This data was developed using the same antibody clone in a different buffer formulation (ab126783).
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All lanes : Anti-Bmi1 antibody [EPR3745(2)] (ab126783) at 1/10000 dilution
Lane 1 : Wild-type MCF7 cell lysate
Lane 2 : BMI1 knockout MCF7 cell lysate
Lane 3 : A431 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 36 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?This data was developed using the same antibody clone in a different buffer formulation (ab126783).
Lanes 1- 3: Merged signal (red and green). Green - ab126783 observed at 37 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.
ab126783 was shown to react with Bmi1 in wild-type MCF7 cells in western blot. Loss of signal was observed when knockout cell line ab262319 (knockout cell lysate ab256851) was used. Wild-type MCF7 and BMI1 knockout MCF7 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab126783 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 10000 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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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling Bmi1 with purified ab126783 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126783).
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Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Bmi1 with purified ab126783 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500) were also used.
Control 1: primary antibody (1/500) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500).
Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126783).
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All lanes : Anti-Bmi1 antibody [EPR3745(2)] (HRP) (ab197620) at 1/1000 dilution
Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : COMMD3-BMI1 knockout HAP1 whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 36 kDaab197620 was shown to recognize Bmi1 in wild-type HAP1 cells as signal was lost at the expected MW in COMMD3-BMI1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and COMMD3-BMI1 knockout samples were subjected to SDS-PAGE. Ab197620 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. The loading control was imaged using the Licor Odyssey CLx prior to blots being developed with ECL technique.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab197620).
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All lanes : Anti-Bmi1 antibody [EPR3745(2)] (ab126783) at 1/1000 dilution
Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : BMI1 knockout HEK293T cell lysate
Lane 3 : MOLT-4 cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 36 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?This data was developed using the same antibody clone in a different buffer formulation (ab126783).
Lanes 1-3: Merged signal (red and green). Green - ab126783 observed at 37 kDa. Red - loading control ab7291 observed at 50 kDa.
ab126783 Anti-Bmi1 antibody [EPR3745(2)] was shown to specifically react with Bmi1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266514 (knockout cell lysate ab256850) was used. Wild-type and Bmi1 knockout samples were subjected to SDS-PAGE. ab126783 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 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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ab126783 (purified) at 1/500 immunoprecipitating Bmi1 in 10 μg K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate (Lanes 1 and 2, observed at 43 kDa). Lane 3 - Rabbit monoclonal IgG (ab172730) instead of ab126783 in K-562 whole cell lysate. For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.
Blocking/Dilution buffer and concentration: 5% NFDM/TBST.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126783).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human normal tonsil tissue labelling Bmi1 with unpurifiied ab126783.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126783).
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colonic adenocarcinoma tissue labelling Bmi1 with unpurifiied ab126783.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126783).
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung adenocarcinoma tissue labelling Bmi1 with unpurifiied ab126783.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126783).
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid gland carcinoma tissue labelling Bmi1 with unpurifiied ab126783.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab126783).
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Protocols
Datasheets and documents
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Datasheet download
Certificate of Compliance
References (5)
ab216444 has been referenced in 5 publications.
- Liang J et al. Bmi-1 regulates the migration and invasion of glioma cells through p16. Cell Biol Int 39:283-90 (2015). PubMed: 25262972
- Dakhova O et al. Genes upregulated in prostate cancer reactive stroma promote prostate cancer progression in vivo. Clin Cancer Res 20:100-9 (2014). PubMed: 24150235
- Wu CP et al. Identification of cancer stem-like side population cells in purified primary cultured human laryngeal squamous cell carcinoma epithelia. PLoS One 8:e65750 (2013). WB ; Human . PubMed: 23776540
- Zhu J et al. Nrf2 is required to maintain the self-renewal of glioma stem cells. BMC Cancer 13:380 (2013). ICC/IF . PubMed: 23937621
- Liu H et al. Single-cell clones of liver cancer stem cells have the potential of differentiating into different types of tumor cells. Cell Death Dis 4:e857 (2013). Flow Cyt ; Human . PubMed: 24136221