Phosphotyrosine anticorps
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- Antigène Tous les produits Phosphotyrosine
- Phosphotyrosine
- Reactivité
- Veuillez nous consulter SVP
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Hôte
- Lapin
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Clonalité
- Polyclonal
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Conjugué
- Cet anticorp Phosphotyrosine est non-conjugé
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Application
- Western Blotting (WB), Immunoprecipitation (IP), Immunofluorescence (IF), Immunohistochemistry (IHC), ELISA, Immunocytochemistry (ICC)
- Specificité
- Detects proteins phosphorylated on tyrosine residues. Does not cross-react with phosphoserine or threonine.
- Purification
- Peptide Affinity Purified
- Immunogène
- Phosphotyrosine conjugated to KLH
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- Indications d'application
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- WB (1:500)
- IHC (1:100)
- ICC/IF (1:50)
- ELISA (1:2000)
- IP (1:100)
- optimal dilutions for assays should be determined by the user.
- Commentaires
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A 1:250 dilution of ABIN361758 was sufficient for detection of tyrosine-phosphorylated species in mouse spleen lysates in western blot analysis.
- Restrictions
- For Research Use only
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- by
- Klinik für Anästhesie, Intensivmedizin und Schmerztherapie, Universitätsklinikum der Ruhr Universität Bochum
- No.
- #103692
- Date
- 29.04.2019
- Antigène
- Phosphotyrosine
- Numéro du lot
- 1508290
- Application validée
- Proximity Ligation Assay
- Contrôle positif
U937 monocytic cells stimulated with ODN1826
- Contrôle négative
Anti pY antibody (ABIN361758) alone
Anti TLR9 antibody (ABIN5542492) alone
- Conclusion
Passed. PLA signal visible in stimulated cells. Negative control with only one primary antibody shows no signal.
- Anticorps primaire
- ABIN361758
- Anticorps secondaire
- ABIN5542492
- Full Protocol
- Grow U937 cells in RPMI (Gibco, 21875-034) supplemented with 10% FCS (Gibco, A3840001) and Pen/Strep(Gibco, 15140-122), at 37°C and 5% CO2 in 500µL in 12-well plates.
- Incubate the cells with 1µM ODN1826 for 4h.
- Use cytospin to spin cells onto slide.
- Fix cells on coverslide in 4% PFA for 20min at RT.
- Wash cells 3x for 5min with PBS.
- Permeabilize cells in PBS containing 0.1% Triton for 5min at RT.
- Block non-specific binding with Duolink PLA Blocking Solution (Sigma-Aldrich) for 30min at RT.
- Incubate cells with primary rabbit anti-pY antibody (antibodies-online, ABIN361758, lot 1508290) diluted 1:100 and primary mouse anti-TLR9 antibody (antibodies-online, ABIN5542492, lot 160127) diluted 1:200 ON at 4°C.
- Wash cells 3x for 5min with PBS.
- Incubate cells with Duolink In Situ PLA Probe anti-Rabbit MINUS (Sigma-Aldrich, DUO92005, lot SLBZ4516) and Duolink In Situ PLA Probe anti-Mouse mouse PLUS (Sigma-Aldrich, DUO92001, lot SLBZ8369) proximity probes according to manufacturers recommendations.
- Wash cells 2x for 5min with PBS.
- Perform ligation step according to the Duolink PLA kit (Sigma).
- Perform amplification step according to the Duolink In Situ PlA Detection Kit Green (Sigma-Aldrich, DUO92014).
- Mount coverslips on glass slides in ProLong Gold antifade reagent (Invitrogen, P36935, lot 1890418) containing DAPI.
- Image acquisition with an Olympus widefield microscope.
- Notes
We used double the amount the Phi29 polymerase for the amplifaction step than recommended.
Validation #103692 (Proximity Ligation Assay)Validation ImagesProtocole -
- Format
- Liquid
- Concentration
- 0.25 mg/mL
- Buffer
- PBS pH 7.0, 50 % glycerol, 0.01 % sodium azide, Storage buffer may change when conjugated
- Agent conservateur
- Sodium azide
- Précaution d'utilisation
- This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
- Stock
- -20 °C
- Stockage commentaire
- -20°C
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- Antigène
- Phosphotyrosine
- Abstract
- Phosphotyrosine Produits
- Classe de substances
- Amino Acid
- Sujet
- Protein phosphorylation is an important posttranslational modification that serves many key functions to regulate a protein's activity, localization, and protein-protein interactions. Phosphorylation is catalyzed by various specific protein kinases, which involves removing a phosphate group from ATP and covalently attaching it to to a recipient protein that acts as a substrate. Most kinases act on both serine and threonine, others act on tyrosine, and a number (dual specificity kinases) act on all three. Because phosphorylation can occur at multiple sites on any given protein, it can therefore change the function or localization of that protein at any time (1). Changing the function of these proteins has been linked to a number of diseases, including cancer, diabetes, heart disease, inflammation and neurological disorders (2-4). In particular, the phosphorylation of tyrosine is considered one of the key steps in signal transduction and regulation of enzymatic activity (5). Phosphotyrosine can be detected through specific antibodies, and are helpful in facilitating the identification of tyrosine kinase substrates (6).
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