Recombinant Anti-Smad2 antibody [EP567Y] - BSA and Azide free (ab216454)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EP567Y] to Smad2 - BSA and Azide free
- Suitable for: ChIC/CUT&RUN-seq, Flow Cyt (Intra), WB, ICC/IF
- Knockout validated
- Reacts with: Mouse, Human
Related conjugates and formulations
Overview
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Product name
Anti-Smad2 antibody [EP567Y] - BSA and Azide free
See all Smad2 primary antibodies -
Description
Rabbit monoclonal [EP567Y] to Smad2 - BSA and Azide free -
Host species
Rabbit -
Specificity
This antibody detects a region about 40AA before the MH2 region (not the MH2 region itself).
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Tested applications
Suitable for: ChIC/CUT&RUN-seq, Flow Cyt (Intra), WB, ICC/IFmore details
Unsuitable for: IHC-P or IP -
Species reactivity
Reacts with: Mouse, Human
Predicted to work with: Rat -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: A549, HeLa and Jurkat cell lysates. ICC/IF: A673 cells. Flow Cyt (intra): Jurkat and PC3 cells. ChIC/CUT&RUN seq: HaCaT cell.
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General notes
ab216454 is the carrier-free version of ab33875.
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
EP567Y -
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 ab216454 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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ChIC/CUT&RUN-seq |
Use at an assay dependent concentration.
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Flow Cyt (Intra) |
Use at an assay dependent concentration.
ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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WB |
Use at an assay dependent concentration. Detects a band of approximately 58 kDa (predicted molecular weight: 58 kDa).
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ICC/IF |
Use at an assay dependent concentration.
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Notes |
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ChIC/CUT&RUN-seq
Use at an assay dependent concentration. |
Flow Cyt (Intra)
Use at an assay dependent concentration. ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
WB
Use at an assay dependent concentration. Detects a band of approximately 58 kDa (predicted molecular weight: 58 kDa). |
ICC/IF
Use at an assay dependent concentration. |
Target
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Function
Receptor-regulated SMAD (R-SMAD) that is an intracellular signal transducer and transcriptional modulator activated by TGF-beta (transforming growth factor) and activin type 1 receptor kinases. Binds the TRE element in the promoter region of many genes that are regulated by TGF-beta and, on formation of the SMAD2/SMAD4 complex, activates transcription. May act as a tumor suppressor in colorectal carcinoma. -
Tissue specificity
Expressed at high levels in skeletal muscle, heart and placenta. -
Sequence similarities
Belongs to the dwarfin/SMAD family.
Contains 1 MH1 (MAD homology 1) domain.
Contains 1 MH2 (MAD homology 2) domain. -
Post-translational
modificationsPhosphorylated on one or several of Thr-220, Ser-245, Ser-250, and Ser-255. In response to TGF-beta, phosphorylated on Ser-465/467 by TGF-beta and activin type 1 receptor kinases. Able to interact with SMURF2 when phosphorylated on Ser-465/467, recruiting other proteins, such as SNON, for degradation. In response to decorin, the naturally occurring inhibitor of TGF-beta signaling, phosphorylated on Ser-240 by CaMK2. Phosphorylated by MAPK3 upon EGF stimulation; which increases transcriptional activity and stability, and is blocked by calmodulin.
In response to TGF-beta, ubiquitinated by NEDD4L; which promotes its degradation.
Acetylated on Lys-19 by coactivators in response to TGF-beta signaling, which increases transcriptional activity. Isoform short: Acetylation increases DNA binding activity in vitro and enhances its association with target promoters in vivo. Acetylation in the nucleus by EP300 is enhanced by TGF-beta. -
Cellular localization
Cytoplasm. Nucleus. Cytoplasmic and nuclear in the absence of TGF-beta. On TGF-beta stimulation, migrates to the nucleus when complexed with SMAD4. On dephosphorylation by phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1. - Information by UniProt
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Database links
- Entrez Gene: 4087 Human
- Entrez Gene: 17126 Mouse
- Entrez Gene: 29357 Rat
- Omim: 601366 Human
- SwissProt: Q15796 Human
- SwissProt: Q62432 Mouse
- SwissProt: O70436 Rat
- Unigene: 12253 Human
see all -
Alternative names
- Drosophila, homolog of, MADR2 antibody
- hMAD-2 antibody
- HsMAD2 antibody
see all
Images
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This data was developed using the same antibody clone in a different buffer formulation (ab33875).
ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/µL, 2.5 x 10^5 HaCaT (Human keratinocyte cell line) cells (treated with 7ng/ml TGF-β for 1h) and 5 µg of ab33875 [EP567Y]. 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. -
All lanes : Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : SMAD2 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 58 kDa
Observed band size: 58 kDaThis data was developed using the same antibody clone in a different buffer formulation (ab33875).
Lanes 1 - 4: Merged signal (red and green). Green - ab33875 observed at 58 kDa. Red - loading control, ab8245 observed at 37 kDa.
ab33875 was shown to react with Smad2 in wild-type HeLa. Loss of signal was observed when knockout cell line ab255430 (knockout cell lysate ab263833) was used. Wild-type and Smad2 knockout samples were subjected to SDS-PAGE. ab33875 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C 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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Overlay histogram showing Jurkat cells stained with purified ab33875 (pink line) at a dilution of 1/110. The cells were fixed with 2% PFA. FITC goat anti-rabbit was used at a dilution of 1/150 and rabbit monoclonal IgG was used as the isotype control (green line).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33875).
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Immunofluorescent staining of A673 cells, fixed with 4% PFA, using purified ab33875 at a dilution of 1/300. An Alexa Fluor® 555 goat anti-rabbit was used at 1/200. The negative control is shown in the bottom right hand panel - for the negative control, purified ab33875 was used at a dilution of 1/200 followed by an Alexa Fluor® 555 goat anti-mouse antibody at a dilution of 1/500.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33875).
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Overlay histogram showing PC3 cells stained with unpurified ab33875 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab33875, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in PC3 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33875).
Protocols
Datasheets and documents
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Datasheet download
Certificate of Compliance
References (6)
ab216454 has been referenced in 6 publications.
- Chen RY et al. Effect of oridonin-mediated hallmark changes on inflammatory pathways in human pancreatic cancer (BxPC-3) cells. World J Gastroenterol 20:14895-903 (2014). WB ; Human . PubMed: 25356049
- Gallo EM et al. Angiotensin II-dependent TGF-ß signaling contributes to Loeys-Dietz syndrome vascular pathogenesis. J Clin Invest 124:448-60 (2014). IF . PubMed: 24355923
- Zieba A et al. Intercellular variation in signaling through the TGF-ß pathway and its relation to cell density and cell cycle phase. Mol Cell Proteomics : (2012). PubMed: 22442258
- Guillory B et al. Lack of fetuin-A (alpha2-HS-glycoprotein) reduces mammary tumor incidence and prolongs tumor latency via the transforming growth factor-beta signaling pathway in a mouse model of breast cancer. Am J Pathol 177:2635-44 (2010). IHC-P ; Mouse . PubMed: 20847285
- Espira L et al. The basic helix-loop-helix transcription factor scleraxis regulates fibroblast collagen synthesis. J Mol Cell Cardiol 47:188-95 (2009). PubMed: 19362560
- Di Bartolo DL et al. KSHV LANA inhibits TGF-beta signaling through epigenetic silencing of the TGF-beta type II receptor. Blood 111:4731-40 (2008). PubMed: 18199825