Anti-CARD9 antibody [RM2079]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CARD9 antibody [RM2079] (AB323568)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labelling CARD9 with ab323568 at 1/50 (9.82 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ 1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in THP-1 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Low expression : A549 (PMID : 32164050).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/ 1000 2ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CARD9 antibody [RM2079] (AB323568)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized A549 (human lung carcinoma epithelial cell, Left) / THP-1 (human monocytic leukemia monocyte, Right) cells labelling CARD9 with ab323568 at 1/500 dilution (0.1 ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Low expression : A549.

• IP

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Immunoprecipitation - Anti-CARD9 antibody [RM2079] (AB323568)

CARD9 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) whole cell lysate with ab323568 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab323568 at 1/1000 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-CARD9 antibody [RM2079] (ab323568) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 10 µg

Lane 2:

ab323568 at 1/30 IP in THP-1 (human monocytic leukemia monocyte) whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab323568 in THP-1 whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 43-62 kDa

false

Exposure time: 10s

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CARD9 antibody [RM2079] (AB323568)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized MEF (mouse embryo fibroblast, Left)/ RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage, Right) cells labelling CARD9 with ab323568 at 1/50 dilution (1 ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Negative control : MEF.

• WB

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Western blot - Anti-CARD9 antibody [RM2079] (AB323568)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : colon (PMID : 11053425).

The expression profile/ molecular weight observed is consistent with what has been described in the literature(PMID : 32164050), This antibody can recognize 3 isoforms of CARD9 and isoforms have different molecular weight.

The identity of the higher MW bands at about 150 kDa and the lower MW bands at about 15 kDa (in lane 1) are unknown

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-CARD9 antibody [RM2079] (ab323568) at 1/1000 dilution

Lane 1:

Human tonsil tissue lysate at 40 µg

Lane 2:

Human colon tissues lysate at 40 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 43-62 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-CARD9 antibody [RM2079] (AB323568)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : A549 (PMID : 32164050 ), 293T (PMID : 33558980).

The expression profile/ molecular weight observed is consistent with what has been described in the literature(PMID : 32164050), This antibody can recognize 3 isoforms of CARD9 and isoforms have different molecular weight.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lane 1 : 81 seconds; Lanes 2-4 : 26 seconds

All lanes:

Western blot - Anti-CARD9 antibody [RM2079] (ab323568) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 2:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 3:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 43-62 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-CARD9 antibody [RM2079] (AB323568)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : MEF.

Low expression : colon (PMID : 11053425).

The expression profile/ molecular weight observed is consistent with what has been described in the literature(PMID : 32164050), This antibody can recognize 3 isoforms of CARD9 and isoforms have different molecular weight.

The identity of the lower MW band at about 30 kDa (in lane 1) is unknown.

The identity of the higher MW bands at about 100 kDa and the lower MW bands at about 40 kDa (in lane 4) are unknown

This blot in lanes 3-4 was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lanes 1-2 : 180 seconds; Lanes 3-5 : 75 seconds

All lanes:

Western blot - Anti-CARD9 antibody [RM2079] (ab323568) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 40 µg

Lane 2:

MEF (mouse embryo fibroblast) whole cell lysate at 40 µg

Lane 3:

Mouse spleen tissue lysate at 40 µg

Lane 4:

Mouse colon lysate at 40 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 43-62 kDa,36 kDa

false