Anti-CXCL5 + CXCL6 antibody [EPR22310-196]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CXCL5 + CXCL6 antibody [EPR22310-196] (AB243097)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A549 (human lung carcinoma cell line) cells labeling CXCL5 + CXCL6 with ab243097 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in A549 cells treated with TNF alpha (10ng/ml 24h), PMA (10nM 24h) and BSA (0.1% 24h) is observed. Tubulin was stained using the Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594, ab195889) at 1/200 dilution (Red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

The expression of CXCL5 and CXCL6 is induced by TNF-a and PMA treatment. (PMID : 9057843; 23922745).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CXCL5 + CXCL6 antibody [EPR22310-196] (AB243097)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized A549 (human lung carcinoma cell line) cell line that was serum starved for 4h, then treated with TNF alpha (10ng/ml 24h), PMA (10nM 24h) and BSA (0.1% 24h) (Red) / Untreated control (Green) labeling CXCL5 + CXCL6with ab243097 at 1/500 dilution, compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488)at 1/2000 dilution was used as the secondary antibody.

The expression of CXCL5 and CXCL6 is induced by TNF-a and PMA. (PMID : 9057843; 23922745).

• WB

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Western blot - Anti-CXCL5 + CXCL6 antibody [EPR22310-196] (AB243097)

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

The expression of CXCL5 and CXCL6 is induced by TNF-a and PMA. (PMID 9057843; PMID : 23922745).

All lanes:

Western blot - Anti-CXCL5 + CXCL6 antibody [EPR22310-196] (ab243097) at 1/500 dilution

Lane 1:

A549 (human lung carcinoma cell line) starved overnight, whole cell lysate at 10 µg

Lane 2:

A549 was starved overnight, then treated with 10ng/ml TNF-a, 10nM Phorbol-12-myristate-13-acetate (PMA) and 0.1% BSA for 24 hours, whole cell lysate at 10 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 1/100000 dilution

Predicted band size: 12 kDa

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Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-CXCL5 + CXCL6 antibody [EPR22310-196] (AB243097)

False colour image of Western blot : Anti-CXCL5 + CXCL6 antibody [EPR22310-196] 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, ab243097 was shown to bind specifically to CXCL5 + CXCL6. A band was observed at 12 kDa in treated wild-type A549 cell lysates with no signal observed at this size in CXCL6 knockout cell line ab275838 (knockout cell lysate ab275812). To generate this image, wild-type and CXCL6 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-CXCL5 + CXCL6 antibody [EPR22310-196] (ab243097) at 1/1000 dilution

Lane 1:

Wild-type A549 Untreated Control cell lysate at 20 µg

Lane 2:

Wild-type A549 Treated TNFa (10 ng/mL, 24 h) + PMA (10 nM, 24 h) cell lysate at 20 µg

Lane 3:

Untreated control at 20 µg

Lane 4:

Treated TNFa (10 ng/mL, 24 h) + PMA (10 nM, 24 h) cell lysate at 20 µg

Lane 5:

Human Lung cell lysate at 20 µg

Lane 6:

MOLT-4 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 12 kDa

Observed band size: 12 kDa

false

• WB

Unknown

Western blot - Anti-CXCL5 + CXCL6 antibody [EPR22310-196] (AB243097)

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

All lanes:

Western blot - Anti-CXCL5 + CXCL6 antibody [EPR22310-196] (ab243097) at 1/1000 dilution

Lane 1:

His-GST-tagged human CXCL5 recombinant protein (aa37-114) 10 ng

Lane 2:

Human CXCL6 recombinant protein (aa40-114) without tag 10 ng

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 1/100000 dilution

Predicted band size: 12 kDa

false

Exposure time: 3s