Recombinant Anti-YTHDF3 antibody [EPR21912-3] (ab220161)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR21912-3] to YTHDF3
- Suitable for: Flow Cyt (Intra), IP, WB, IHC-Fr, IHC-P
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-YTHDF3 antibody [EPR21912-3]
See all YTHDF3 primary antibodies -
Description
Rabbit monoclonal [EPR21912-3] to YTHDF3 -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt (Intra), IP, WB, IHC-Fr, IHC-Pmore details
Unsuitable for: ICC/IF -
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: Wild-type mESC whole cell lysate. GST-tagged human YTHDF3 recombinant protein. Fresh HT-1080, HeLa, NIH/3T3, PC-12 whole cell lysate. IHC-P: Mouse and rat cerebrum tissue. Human colon tissue. IHC-Fr: Mouse and rat cerebrum tissue. Flow Cyt (intra): HeLa cells. IP: HeLa whole cell lysate.
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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.
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: 40% Glycerol (glycerin, glycerine), 0.05% BSA, PBS -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR21912-3 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Immunohistochemistry reagents
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Isotype control
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab220161 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) |
1/60.
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IP |
1/30.
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WB | (1) |
1/1000. Detects a band of approximately 73 kDa (predicted molecular weight: 64 kDa).
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IHC-Fr |
1/100.
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IHC-P | (1) |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Use at 1/500 (mouse, rat) and 1/100 (human) dilution. |
Notes |
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Flow Cyt (Intra)
1/60. |
IP
1/30. |
WB
1/1000. Detects a band of approximately 73 kDa (predicted molecular weight: 64 kDa). |
IHC-Fr
1/100. |
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. Use at 1/500 (mouse, rat) and 1/100 (human) dilution. |
Target
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Function
Specifically recognizes and binds N6-methyladenosine (m6A)-containing RNAs. M6A is a modification present at internal sites of mRNAs and some non-coding RNAs and plays a role in the efficiency of mRNA splicing, processing and stability. -
Sequence similarities
Contains 1 YTH domain. - Information by UniProt
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Database links
- Entrez Gene: 253943 Human
- Entrez Gene: 229096 Mouse
- Entrez Gene: 361920 Rat
- SwissProt: Q7Z739 Human
- SwissProt: Q8BYK6 Mouse
- Unigene: 491861 Human
- Unigene: 23834 Mouse
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Alternative names
- FLJ31657 antibody
- YTH domain family 3 antibody
- YTH domain family member 3 antibody
see all
Images
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All lanes : Anti-YTHDF3 antibody [EPR21912-3] (ab220161) at 1/1000 dilution
Lane 1 : Wild-type mESC (mouse embryo stem cell) whole cell lysate
Lane 2 : YTHDF3 knockout mESC 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: 64 kDa
Observed band size: 73 kDa why is the actual band size different from the predicted?
Exposure time: 59 secondsThe wild-type and YTHDF3 knockout cell lysates were kindly provided by an anonymous collaborator.
ab220161 was shown to specifically react with YTHDF3 in wild-type mESC cells as signal was lost in YTHDF3 knockout cells. Wild-type and YTHDF3 knockout samples were subjected to SDS-PAGE. ab220161 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.
The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.
Blocking/Dilution buffer: 5% NFDM/TBST.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YTHDF3 antibody [EPR21912-3] (ab220161)
Immunohistochemical analysis of paraffin-embedded human colon tissue labeling YTHDF3 with ab220161 at 1/100 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on human colon (PMID:29103884, 28250115) is observed. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).
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Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum tissue labeling YTHDF3 with ab220161 at 1/100 dilution (green), followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Cytoplasmic and nuclear staining in mouse cerebrum (PMID:29103884, 28250115) is observed. Counterstained with DAPI (blue).
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit used at a 1/1,000 dilution.
Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).
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Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cell line labeling YTHDF3 with ab220161 at 1/60 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.
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YTHDF3 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate with ab220161 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab220161 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used at 1/5000 dilution.
Lane 1: HeLa whole cell lysate 10 µg (Input).
Lane 2: ab220161 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab220161 in HeLa whole cell lysate.Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 15 seconds.Lysate were made freshly and used in IP test immediately to minimize protein degradation. Incubation time was 2h.
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All lanes : Anti-YTHDF3 antibody [EPR21912-3] (ab220161) at 1/1000 dilution
Lane 1 : HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate (fresh lysate)
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate (fresh lysate)
Lane 3 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate (fresh lysate)
Lane 4 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate (fresh lysate)
Lane 5 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 6 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 7 : PC-12 (rat adrenal gland pheochromocytoma) 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: 64 kDaLysate should be made freshly and used in WB immediately to minimize protein degradation (lane1-4). Lane 5-7 are the lysates from same cell lines but have experienced freeze-thaw cycles.
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure times: Lanes 1-4: 37 secs; Lanes 5-7: 92 secs.
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All lanes : Anti-YTHDF3 antibody [EPR21912-3] (ab220161) at 1/5000 dilution
Lane 1 : GST-tagged human YTHDF1 recombinant protein 20 ng
Lane 2 : GST-tagged human YTHDF2 recombinant protein 20 ng
Lane 3 : GST-tagged human YTHDF3 recombinant protein 20 ng
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 64 kDa
Exposure time: 10 secondsThe YTHDF recombinant proteins were kindly provided by an anonymous collaborator.
Blocking/Dilution buffer: 5% NFDM/TBST.
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Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebrum tissue labeling YTHDF3 with ab220161 at 1/100 dilution (green), followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Cytoplasmic and nuclear staining in rat cerebrum (PMID:29103884, 28250115) is observed. Counterstained with DAPI (blue).
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit used at a 1/1,000 dilution.
Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YTHDF3 antibody [EPR21912-3] (ab220161)
Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling YTHDF3 with ab220161 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on neurons of rat cerebrum (PMID:29103884, 28250115) is observed. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YTHDF3 antibody [EPR21912-3] (ab220161)
Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling YTHDF3 with ab220161 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on neurons of mouse cerebrum (PMID:29103884, 28250115) is observed. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).
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
Certificate of Compliance
References (9)
ab220161 has been referenced in 9 publications.
- Sun Z et al. RNA demethylase ALKBH5 inhibits TGF-ß-induced EMT by regulating TGF-ß/SMAD signaling in non-small cell lung cancer. FASEB J 36:e22283 (2022). WB ; Human . PubMed: 35344216
- You A et al. TTC22 promotes m6A-mediated WTAP expression and colon cancer metastasis in an RPL4 binding-dependent pattern. Oncogene N/A:N/A (2022). WB ; Human . PubMed: 35798874
- Tao L et al. FTO modifies the m6A level of MALAT and promotes bladder cancer progression. Clin Transl Med 11:e310 (2021). PubMed: 33634966
- Xu H et al. YTHDF2 alleviates cardiac hypertrophy via regulating Myh7 mRNA decoy. Cell Biosci 11:132 (2021). PubMed: 34266473
- Ding H et al. GNAS promotes inflammation-related hepatocellular carcinoma progression by promoting STAT3 activation. Cell Mol Biol Lett 25:8 (2020). WB ; Human . PubMed: 32123532
- Jurczyszak D et al. HIV protease cleaves the antiviral m6A reader protein YTHDF3 in the viral particle. PLoS Pathog 16:e1008305 (2020). WB ; Human . PubMed: 32053707
- Jin D et al. m6A demethylase ALKBH5 inhibits tumor growth and metastasis by reducing YTHDFs-mediated YAP expression and inhibiting miR-107/LATS2-mediated YAP activity in NSCLC. Mol Cancer 19:40 (2020). PubMed: 32106857
- Jian D et al. METTL14 aggravates endothelial inflammation and atherosclerosis by increasing FOXO1 N6-methyladeosine modifications. Theranostics 10:8939-8956 (2020). PubMed: 32802173
- Jin D et al. m6A mRNA methylation initiated by METTL3 directly promotes YAP translation and increases YAP activity by regulating the MALAT1-miR-1914-3p-YAP axis to induce NSCLC drug resistance and metastasis. J Hematol Oncol 12:135 (2019). WB ; Human . PubMed: 31818312