Anti-IL-6 antibody [EPR22565-204]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-IL-6 antibody [EPR22565-204] (AB233551)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HUVEC (human umbilical vein endothelial cell) cells labeling IL-6 with ab233551 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 HUVEC cells treated with lipopolysaccharide (0.5μg/ml) for 4 h, then together with Brefeldin A (300ng/ml) for another 20h. The nuclear counterstain is DAPI (blue). Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red). The negative control is the secondary antibody only.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-IL-6 antibody [EPR22565-204] (AB233551)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HUVEC (Human umbilical vein endothelial cell) treated with 0.5ug/ml LPS for 4h, then together with 300ng/ml BFA for another 20h (Red) / Untreated control (Green), labeling IL-6 with ab233551 at 1/400 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IL-6 antibody [EPR22565-204] (AB233551)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) (top) or PBMCs treated with LPS, 18 Hours, 1µg/mL (bottom), with ab233551 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left).

PBMCs were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by incubation with the CD14 multistain. Cells were then fixed and permeabilised with BD Cytofix/Cytoperm™ for 20 min, and stained with the antibody ab233551 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 0.04 μg/ml (1/12)) for 30 min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C.

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.

• IP

Unknown

Immunoprecipitation - Anti-IL-6 antibody [EPR22565-204] (AB233551)

IL-6 was immunoprecipitated from 0.35 mg HUVEC (Human umbilical vein endothelial cell) treated with 0.5μg/ml LPS for 4h, then together with 300ng/ml BFA for another 20h whole cell lysate with ab233551 at 1/20 dilution. Western blot was performed from the immunoprecipitate using ab233551 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used at 1/5000 dilution.

Lane 1 : HUVEC treated as above whole cell lysate 10 μg (Input).

Lane 2 : ab233551 IP in HUVEC treated as above whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab233551 in HUVEC treated as above whole cell lysate.

Blocking/Dilution buffer : 5% NFDM/TBST.
Exposure time : 15 seconds.

All lanes:

Immunoprecipitation - Anti-IL-6 antibody [EPR22565-204] (ab233551)

Predicted band size: 23 kDa

Observed band size: 21 kDa,28 kDa

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• WB

Lab

Western blot - Anti-IL-6 antibody [EPR22565-204] (AB233551)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

In Western blot analysis, ab233706 exhibited lower sensitivity compared to ab233551, for which reason we recommend the latter one as superior alternative.

ab233706 does not work in THP-1 cell line due to sensitivity issue.

Lanes 1 - 2:

Western blot - Anti-IL-6 antibody [EPR21711] (<a href='/en-us/products/primary-antibodies/il-6-antibody-epr21711-ab233706'>ab233706</a>) at 1/1000 dilution

Lanes 3 - 4:

Western blot - Anti-IL-6 antibody [EPR22565-204] (ab233551) at 1/1000 dilution

Lanes 1 and 3:

Untreated HUVEC (human umbilical vein endothelial cell line) whole cell lysate at 20 µg

Lanes 2 and 4:

HUVEC treated with 0.5 ug/ml lipopolysaccharides (LPS) for 24 hours, added 300 ng/ml BFA last 20 hours 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: 23 kDa

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Exposure time: 20s

• WB

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Western blot - Anti-IL-6 antibody [EPR22565-204] (AB233551)

The molecular weight observed is consistent with what has been described in the literature (PMID : 14970177).

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-IL-6 antibody [EPR22565-204] (ab233551) at 1/1000 dilution

Lane 1:

Untreated HUVEC (human umbilical vein endothelial cell) whole cell lysate at 20 µg

Lane 2:

HUVEC treated with 0.5 μg/ml LPS for 4 hours, then 300 ng/ml BFA (Brefeldin A) was added for the last 20 hours, 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: 23 kDa

Observed band size: 21 kDa,24 kDa

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• WB

Lab

Western blot - Anti-IL-6 antibody [EPR22565-204] (AB233551)

False colour image of Western blot : Anti-IL-6 antibody [EPR22565-204] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab233551 was shown to bind specifically to IL-6. A band was observed at 25 kDa in wild-type A549 cell lysates with no signal observed at this size in IL6 knockout cell line ab273751 (knockout cell lysate ab275501). To generate this image, wild-type and IL6 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 fluorescent western blot (TBS-based) blocking solution 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 1/20000 dilution.

All lanes:

Western blot - Anti-IL-6 antibody [EPR22565-204] (ab233551) at 1/1000 dilution

Lane 1:

Wild-type A549 Vehicle Control IL-1b (0 ng/mL, 24 h), Brefeldin A (5 ug/mL, final 6 h) cell lysate at 30 µg

Lane 2:

Wild-type A549 Treated IL-1b (20 ng/mL, 24 h), Brefeldin A (5 ug/mL, final 6 h) cell lysate at 30 µg

Lane 3:

IL-6 knockout A549 Vehicle Control IL-1b (0 ng/mL, 24 h), Brefeldin A (5 ug/mL, final 6 h) cell lysate at 30 µg

Lane 4:

IL-6 knockout A549 Treated IL-1b (20 ng/mL, 24 h), Brefeldin A (5 ug/mL, final 6 h) cell lysate at 30 µg

Predicted band size: 23 kDa

Observed band size: 25 kDa

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