Phosphotyrosine anticorps

Détails pour le produit réf. ABIN361758, Fournisseur: Connectez-vous pour afficher
Antigène
Reactivité
Toutes les espèces
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58
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10
3
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1
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Hôte
Lapin
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3
Clonalité
Polyclonal
Conjugué
Inconjugué
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28
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2
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2
2
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2
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2
1
1
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1
Application
ELISA, Immunocytochemistry (ICC), Immunofluorescence (IF), Immunohistochemistry (IHC), Immunoprecipitation (IP), Western Blotting (WB)
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131
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65
42
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24
13
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Options
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N° du produit (Fournisseur)
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'Independent Validation' signe
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
Protocole
  • 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 Images
Proximity Ligation Assay validation image for anti-Phosphotyrosine antibody (ABIN361758) Negative controls of U937 cells without either ABIN361758 or ABIN5542492 show no sign...
Immunogène Phosphotyrosine conjugated to KLH
Specificité Detects proteins phosphorylated on tyrosine residues. Does not cross-react with phosphoserine or threonine.
Purification Peptide Affinity Purified
Antigène
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).
Indications d'application
  • 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

A 1:250 dilution of SPC-161 was sufficient for detection of tyrosine-phosphorylated species in mouse spleen lysates in western blot analysis.

Restrictions For Research Use only
Format Liquid
Concentration 0.25 mg/mL
Buffer PBS pH 7.0, 50 % glycerol, 0.01 % sodium azide
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
Images (Fournisseur)
Immunofluorescence (fixed cells) (IF/ICC) image for anti-Phosphotyrosine antibody (ABIN361758) Immunocytochemistry/Immunofluorescence analysis using Rabbit Anti-Phosphotyrosine Pol...
Immunofluorescence (fixed cells) (IF/ICC) image for anti-Phosphotyrosine antibody (ABIN361758) Immunocytochemistry/Immunofluorescence analysis using Rabbit Anti-Phosphotyrosine Pol...
Immunohistochemistry (IHC) image for anti-Phosphotyrosine antibody (ABIN361758) Immunohistochemistry analysis using Rabbit Anti-Phosphotyrosine Polyclonal Antibody ....
Background publications Goto, Kiyono, Tomono, Kawajiri, Urano, Furukawa, Nigg, Inagaki: "Complex formation of Plk1 and INCENP required for metaphase-anaphase transition." dans: Nature cell biology, Vol. 8, Issue 2, pp. 180-7, 2006 (PubMed).

Blume-Jensen, Hunter: "Oncogenic kinase signalling." dans: Nature, Vol. 411, Issue 6835, pp. 355-65, 2001 (PubMed).

Downward: "The ins and outs of signalling." dans: Nature, Vol. 411, Issue 6839, pp. 759-62, 2001 (PubMed).

Pawson, Saxton: "Signaling networks--do all roads lead to the same genes?" dans: Cell, Vol. 97, Issue 6, pp. 675-8, 1999 (PubMed).

Ostrovsky, Maloy: "Protein phosphorylation on serine, threonine, and tyrosine residues modulates membrane-protein interactions and transcriptional regulation in Salmonella typhimurium." dans: Genes & development, Vol. 9, Issue 16, pp. 2034-41, 1995 (PubMed).

Frackelton, Ross, Eisen: "Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells." dans: Molecular and cellular biology, Vol. 3, Issue 8, pp. 1343-52, 1983 (PubMed).

Ross, Baltimore, Eisen: "Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten." dans: Nature, Vol. 294, Issue 5842, pp. 654-6, 1982 (PubMed).

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