TLR9 anticorps (AA 868-1016)

N° du produit ABIN5542492

Détail du produit anti-TLR9 anticorps

Antigène: TLR9 (Toll-Like Receptor 9 (TLR9))

Épitope: AA 868-1016

Reactivité: Humain

Hôte: Souris

Clonalité: Monoclonal

Conjugué: Cet anticorp TLR9 est non-conjugé

Application: Western Blotting (WB), Immunohistochemistry (IHC), ELISA, Flow Cytometry (FACS), Immunocytochemistry (ICC)

Purification: purified

Immunogène: Purified recombinant fragment of human TLR9 (AA: 868-1016) expressed in E. coli.

Clone: 1B12H2

Isotype: IgG2a

Information d'application

Indications d'application: ELISA: 1:10000, WB: 1:500 - 1:2000, IHC: 1:200 - 1:1000, ICC: 1:200 - 1:1000, FCM: 1:200 - 1:400

Restrictions: For Research Use only

Independent Validation (IF)

Validation #103693 (Immunofluorescence)

by: Klinik für Anästhesie, Intensivmedizin und Schmerztherapie, Universitätsklinikum der Ruhr Universität Bochum

No.: #103693

Date: 26.03.2019

Antigène: TLR9

Numéro du lot: 160127

Application validée: Immunofluorescence

Contrôle positif:

U937 monocytic cells

Contrôle négative:

No primary antibody control

Conclusion:

Passed. ABIN5542492 localization on ER is visible. Negative control without primary antibody shows no signal.

Validation Images

U937 monocytic cells stained with with ABIN5542492 (top row). TLR9 staining (AF488, green) shows typical peri-nuclear localization (ER). Counterstain with DAPI (blue). Negative control without primary antibody (bottom row) shows no significant staining. Counterstain with DAPI (blue).

Negative controls of U937 cells without either ABIN5542492 or ABIN361758 show no signal. In U937 cells incubated simulatnously with ABIN5542492 and ABIN361758 the PLA signal (green) is clearly visible. Counterstain with DAPI (blue).

Protocole

Anticorps primaire: ABIN5542492

Anticorps secondaire: AF488-conjugated AffiniPure alpaca anti-Mouse IgG antibody (Jackson ImmunoResearch, 615-545-214)

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.
• 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) for 30 min at RT.
• Incubate cells with primary mouse anti-TLR9 antibody (antibodies-online, ABIN5542492, lot 160127) diluted 1:200 in staining solution ON at 4°C.
• Wash cells 3x for 5min with PBS.
• Incubate cells with secondary AF488-conjugated AffiniPure alpaca anti-Mouse IgG antibody (Jackson ImmunoResearch, 615-545-214) diluted 1:400 in staining solution for 1h at RT.
• Wash cells 2x for 5min with PBS.
• Mount coverslips on glass slides in ProLong Gold antifade reagent (Invitrogen, P36935,1890418) containing DAPI.
• Image acquisition with an Olympus widefield microscope.

Notes:

TLR9 staining using ABIN5542492 shows typical localization peri-nuclear localization.

Independent Validation (PLA)

Validation #103821 (Proximity Ligation Assay)

by: Klinik für Anästhesie, Intensivmedizin und Schmerztherapie, Universitätsklinikum der Ruhr Universität Bochum

No.: #103821

Date: 29.04.2019

Antigène: TLR9

Numéro du lot: 160127

Application validée: Proximity Ligation Assay

Contrôle positif:

U937 monocytic cells stimulated with ODN1826

Contrôle négative:

Anti TLR9 antibody (ABIN5542492) alone

Anti pY antibody (ABIN361758) alone

Conclusion:

Passed. PLA signal visible in stimulated cells. Negative control with only one primary antibody shows no signal.

Validation Images

U937 monocytic cells stained with with ABIN5542492 (top row). TLR9 staining (AF488, green) shows typical peri-nuclear localization (ER). Counterstain with DAPI (blue). Negative control without primary antibody (bottom row) shows no significant staining. Counterstain with DAPI (blue).

Negative controls of U937 cells without either ABIN5542492 or ABIN361758 show no signal. In U937 cells incubated simulatnously with ABIN5542492 and ABIN361758 the PLA signal (green) is clearly visible. Counterstain with DAPI (blue).

Protocole

Anticorps primaire: ABIN5542492

Anticorps secondaire: ABIN361758

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 mouse anti-TLR9 antibody (antibodies-online, ABIN5542492, lot 160127) diluted 1:200 and primary rabbit anti-pY antibody (antibodies-online, ABIN361758, lot 1508290) diluted 1:100 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.

Stockage

Format: Liquid

Buffer: Purified antibody in PBS with 0.05 % 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: 4 °C/-20 °C

Stockage commentaire: 4°C, -20°C for long term storage

Détails sur TLR9

Antigène: TLR9 (Toll-Like Receptor 9 (TLR9))

Autre désignation: TLR9 (TLR9 Produits)

Synonymes: anticorps CD289, anticorps toll like receptor 9, anticorps toll-like receptor 9, anticorps TLR9, anticorps Tlr9

Sujet:

Description: The protein encoded by this gene is a member of the Toll-like receptor (TLR) family which plays a fundamental role in pathogen recognition and activation of innate immunity. TLRs are highly conserved from Drosophila to humans and share structural and functional similarities. They recognize pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, and mediate the production of cytokines necessary for the development of effective immunity. The various TLRs exhibit different patterns of expression. This gene is preferentially expressed in immune cell rich tissues, such as spleen, lymph node, bone marrow and peripheral blood leukocytes. Studies in mice and human indicate that this receptor mediates cellular response to unmethylated CpG dinucleotides in bacterial DNA to mount an innate immune response.,

Aliases: CD289

Poids moléculaire: 115.8 kDa

ID gène: 54106

HGNC: 54106

Pathways: Signalisation TLR, Activation of Innate immune Response, Cellular Response to Molecule of Bacterial Origin, Toll-Like Receptors Cascades