TLR9 antibody (AA 868-1016)

Catalog No. ABIN5542492

This anti-TLR9 antibody is a Mouse Monoclonal antibody detecting TLR9 in IHC, ELISA, FACS and ICC. Suitable for Human. Independently validated for use in Immunofluorescence, Proximity Ligation Assay.

Quick Overview for TLR9 antibody (AA 868-1016) (ABIN5542492)

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

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This TLR9 antibody is un-conjugated

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

Clone: 1B12H2

Product Details anti-TLR9 Antibody

Binding Specificity: AA 868-1016

Purpose: TLR9 Antibody

Purification: Purified antibody

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

Isotype: IgG2a

Application Details

Application Notes:

ELISA: 1/10000

FCM: 1/200 - 1/400

ICC: 1/200 - 1/1000

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: 03/26/2019

Antigen: TLR9

Lot Number: 160127

Method validated: Immunofluorescence

Positive Control:

U937 monocytic cells

Negative Control:

No primary antibody control

Notes:

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).

Full Methods

Primary Antibody: ABIN5542492

Secondary Antibody: 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.

Experimental 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: 04/29/2019

Antigen: TLR9

Lot Number: 160127

Method validated: Proximity Ligation Assay

Positive Control:

U937 monocytic cells stimulated with ODN1826

Negative Control:

Anti TLR9 antibody (ABIN5542492) alone

Anti pY antibody (ABIN361758) alone

Notes:

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

Validation Images

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).

Full Methods

Primary Antibody: ABIN5542492

Secondary Antibody: 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.

Experimental Notes:

We used double the amount the Phi29 polymerase for the amplifaction step than recommended.

Handling

Format: Liquid

Buffer: Purified antibody in PBS with 0.05 % sodium azide.

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Storage Comment: Store at 4°C short term. Aliquot and store at -20°C long term. Avoid freeze/thaw cycles.

Target Details for TLR9

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

Alternative Name: TLR9

Background: 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.

Molecular Weight: 115.8 kDa

UniProt: Q9NR96

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