Anti-IL-8 antibody [EPR26511-74]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-IL-8 antibody [EPR26511-74] (AB289967)

Intracellular flow cytometric analysis of 2% paraformaldehyde fixed, 0.1% saponin permeabilised human peripheral blood mononuclear cell (PBMC) treated with 1μg/ml Lipopolysaccharide (LPS) for 22 hours, then add 3uM Monensin for another 2h (Right). Untreated control (Left).

Primary antibody : ab289967, at 1/500 dilution.

Secondary antibody : Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution.

Scatter image shows specific IL-8 expression in LPS induced monocyte population.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IL-8 antibody [EPR26511-74] (AB289967)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U-937 cells labelling IL-8 with ab289967 at 1/100 (5.87 μg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 μg/mL) dilution (Green). Confocal image showing cytoplasmic staining is observed in U-937 cells treated with TPA (100 ng/mL) for 24 h, then LPS (5 μg/mL) for 7 h with Brefeldin A (300 ng/mL) for the last 3 h. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5μg/ml) dilution (Red). 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 (2 μg/mL) dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-IL-8 antibody [EPR26511-74] (AB289967)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized U-937 (Human histiocytic lymphoma monocyte) treated with 100ng/ml TPA for 24 hours, then 5μg/ml LPS for 4 hours, and add 300ng/ml BFA for another 3h (Red) / Untreated control (Green) cells labelling IL-8 with ab289967 at 1/500 dilution (0.1μg) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-IL-8 antibody [EPR26511-74] (AB289967)

IL-8 was immunoprecipitated from U937 (human histiocytic lymphoma monocyte) treated with 100ng/ml TPA for 24h then treated with 5 μg/ml LPS for 4h, and add 300 ng/ml BFA for another 3h, whole cell lysate with ab289967 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab289967 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : U937 (human histiocytic lymphoma monocyte) treated with 100 ng/ml TPA for 24h then treated with 5 μg/ml LPS for 4h, and add 300 ng/ml BFA for another 3h, whole cell lysate 10 μg

Lane 2 : ab289967 IP in U937 treated with 100 ng/ml TPA for 24h then treated with 5 μg/ml LPS for 4h, and add 300 ng/ml BFA for another 3h, whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab289967 in U937 treated with 100 ng/ml TPA for 24h then treated with 5 μg/ml LPS for 4h, and add 300 ng/ml BFA for another 3h, whole cell lysate

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

Exposure time : 50 seconds

All lanes:

Immunoprecipitation - Anti-IL-8 antibody [EPR26511-74] (ab289967)

Predicted band size: 11 kDa

false

• WB

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Western blot - Anti-IL-8 antibody [EPR26511-74] (AB289967)

Blocking buffer and concentration was Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Diluting buffer was Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBST.

Lysates/proteins at 20 μg per lane.
Performed under reducing conditions.
False colour image of Western blot : Anti-IL-8 antibody [EPR26511-74] (ab289967) 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, ab289967 was shown to bind specifically to IL-8. A band was observed at 11 kDa in wild-type PC-3 cell lysates with no signal observed at this size in CXCL8 knockout cell lysates. To generate this image, wild-type and CXCL8 knockout PC-3 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/10000 dilution.

All lanes:

Western blot - Anti-IL-8 antibody [EPR26511-74] (ab289967) at 1/1000 dilution

Lane 1:

Wild-type PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Wild-type PC-3 treated with 2μg/ml LPS for 5h, then treated with 5μg/ml Brefeldin A for 5h, whole cell lysate at 20 µg

Lane 3:

CXCL8 knockout PC-3 whole cell lysate at 20 µg

Lane 4:

CXCL8 knockout PC-3 treated with 2μg/ml LPS for 5h, then treated with 5μg/ml Brefeldin A for 5h, whole cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Lanes 1 - 4:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 11 kDa

Observed band size: 11 kDa

false

• WB

Unknown

Western blot - Anti-IL-8 antibody [EPR26511-74] (AB289967)

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

All lanes:

Western blot - Anti-IL-8 antibody [EPR26511-74] (ab289967) at 1/1000 dilution

Lane 1:

Untreated U-937 (human histiocytic lymphoma monocyte) whole cell lysate at 20 µg

Lane 2:

U-937 treated with TPA (100ng/mL) for 24 h, then treated with LPS (5 µg/mL) for 7 h with Brefeldin A (300 ng/mL) for the last 3 h, whole cell lysate at 20 µg

Lane 3:

Untreated U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

U-87 MG treated with 1μM Thapsigargin for 24h, 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 1/100000 dilution

Predicted band size: 11 kDa

Observed band size: 11 kDa

false

Exposure time: 70s