Anti-CXCL9 antibody [EPR23999-5]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CXCL9 antibody [EPR23999-5] (AB263442)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized THP-1 (human monocytic leukemia monocyte) treated with 100ng/ml IFN-gamma and 1ug/ml LPS (Lipopolysaccharide) for 48 hours (Red)/ Untreated control (Green) cells labelling CXCL9 with ab263442 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-CXCL9 antibody [EPR23999-5] (AB263442)

CXCL9 was immunoprecipitated from 0.35 mg THP-1(human monocytic leukemia monocyte) (treated with 100ng/ml IFN-γ and 1ug/ml LPS (Lipopolysaccharide) for 48 hours) whole cell lysate with ab263442 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab263442 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : THP-1 treated with 100ng/ml IFN-γ and 1ug/ml LPS (Lipopolysaccharide) for 48 hours, whole cell lysate 10 ug

Lane 2 : ab263442 IP in THP-1 treated with 100ng/ml IFN-γ and 1ug/ml LPS(Lipopolysaccharide) for 48 hours whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab263442 in THP-1 treated with 100ng/ml IFN-γ and 1ug/ml LPS(Lipopolysaccharide) for 48 hours whole cell lysate

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-CXCL9 antibody [EPR23999-5] (ab263442)

Predicted band size: 14 kDa

Observed band size: 13 kDa,14 kDa

false

• WB

Lab

Western blot - Anti-CXCL9 antibody [EPR23999-5] (AB263442)

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

The expression profile is consistent with what has been described in the literature (PMID : 15154616, 22973274).

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-CXCL9 antibody [EPR23999-5] (ab263442) at 1/1000 dilution

Lane 1:

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

Lane 2:

THP-1 (treated with 200 ng /ml IFN-gamma and 50 ng/ml LPS (Lipopolysaccharide) for 24 hours), whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 14 kDa

Observed band size: 13 kDa,14 kDa

false

• WB

Lab

Western blot - Anti-CXCL9 antibody [EPR23999-5] (AB263442)

False colour image of Western blot : Anti-CXCL9 antibody [EPR23999-5] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab263442 was shown to bind specifically to CXCL9. A band was observed at 17 kDa in treated wild-type THP-1 cell lysates with no signal observed at this size in CXCL9 knockout cell line ab282347 (knockout cell lysate ab283019). To generate this image, wild-type and CXCL9 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CXCL9 antibody [EPR23999-5] (ab263442) at 1/1000 dilution

Lane 1:

Wild-type THP-1 untreated control cell lysate at 20 µg

Lane 2:

Wild-type THP-1 treated IFNg (200 ng/mL, 24 h) + LPS (50 ng/mL, 24 h) cell lysate at 20 µg

Lane 3:

CXCL9 knockout THP-1 untreated control cell lysate at 20 µg

Lane 4:

CXCL9 knockout THP-1 treated IFNg (200 ng/mL, 24 h) + LPS (50 ng/mL, 24 h) cell lysate at 20 µg

Observed band size: 17 kDa

false

• WB

Lab

Western blot - Anti-CXCL9 antibody [EPR23999-5] (AB263442)

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

The expression profile is consistent with what has been described in the literature (PMID : 15154616, 22973274).

Exposure time : 5.5 seconds.

All lanes:

Western blot - Anti-CXCL9 antibody [EPR23999-5] (ab263442) at 1/1000 dilution

All lanes:

His-tagged human purified recombinant full-length protein CXCL9, 10 ng

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 14 kDa

Observed band size: 13 kDa,14 kDa

false