Recombinant Anti-IL-8 antibody [EPR26511-74] - BSA and Azide free (ab289992)

All lanes : 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
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
Lane 3 : CXCL8 knockout PC-3 whole cell lysate
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

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 11 kDa
Observed band size: 11 kDa

This data was developed using ab289967, the same antibody clone in a different buffer formulation. 

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

Lane 1 : Untreated U-937 (human histiocytic lymphoma monocyte) whole cell lysate
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
Lane 3 : Untreated U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate
Lane 4 : U-87 MG treated with 1µM Thapsigargin for 24h, whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 11 kDa
Observed band size: 11 kDa


Exposure time: 70 seconds

This data was developed using ab289967, the same antibody clone in a different buffer formulation. 

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

This data was developed using ab289967, the same antibody clone in a different buffer formulation.

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.

This data was developed using ab289967, the same antibody clone in a different buffer formulation.

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.

This data was developed using ab289967, the same antibody clone in a different buffer formulation. 

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.

This data was developed using ab289967, the same antibody clone in a different buffer formulation.

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