Anti-IL-6 antibody [EPR21711]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IL-6 antibody [EPR21711] (AB233706)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HUVEC (human umbilical vein endothelial cell line) cells labeling IL6 with ab233706 at 1/50 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 (0.5 μg/ml) for 24h and Brefeldin A (300 ng/ml) for 20h. The nuclear counterstain is DAPI (blue). Tubulin is detected with 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-6 antibody [EPR21711] (AB233706)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HUVEC (human umbilical vein endothelial cell line) treated with lipopolysaccharide (0.5 μg/ml) for 24h and Brefeldin A (300 ng/ml) for 20h (red) / untreated control (green) cells labeling IL6 with ab233706 at 1/500 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.

• IP

Supplier Data

Immunoprecipitation - Anti-IL-6 antibody [EPR21711] (AB233706)

IL6 was immunoprecipitated from 0.35 mg HUVEC (human umbilical vein endothelial cell line) treated with lipopolysaccharide (0.5 μg/ml) for 24h and Brefeldin A (300 ng/ml) for 20h, whole cell lysate with ab233706 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab233706 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HUVEC treated with lipopolysaccharide (0.5 μg/ml) for 24h and Brefeldin A (300 ng/ml) for 20h, whole cell lysate 10 μg (Input).
Lane 2 : ab233706 IP in HUVEC treated with lipopolysaccharide (0.5 μg/ml) for 24h and Brefeldin A (300 ng/ml) for 20h, whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab233706 in HUVEC treated with lipopolysaccharide (0.5 μg/ml) for 24h and Brefeldin A (300 ng/ml) for 20h, whole cell lysate.

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

All lanes:

Immunoprecipitation - Anti-IL-6 antibody [EPR21711] (ab233706)

Predicted band size: 23 kDa

false

• WB

Lab

Western blot - Anti-IL-6 antibody [EPR21711] (AB233706)

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] (ab233706) at 1/1000 dilution

Lanes 3 - 4:

Western blot - Anti-IL-6 antibody [EPR22565-204] (<a href='/en-us/products/primary-antibodies/il-6-antibody-epr22565-204-ab233551'>ab233551</a>) 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

false

Exposure time: 20s

• WB

Lab

Western blot - Anti-IL-6 antibody [EPR21711] (AB233706)

Lanes 1 - 4 : Merged signal (red and green). Green - ab233706 observed at 25 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab233706 was shown to react with IL-6 in wild-type A549 cells in western blot with loss of signal observed in IL-6 knockout cell line ab273751 (knockout cell lysate ab275501). Wild-type and IL-6 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab233706 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-IL-6 antibody [EPR21711] (ab233706) at 1/1000 dilution

Lane 1:

Wild-type A549 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 4h) cell lysate at 30 µg

Lane 2:

Wild-type A549 IL-1β (<a href='/en-us/products/proteins-peptides/recombinant-human-il-1-beta-protein-active-ab259387'>ab259387</a>) (20 ng/ml, 24h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5 ug/ml for the last 4h) cell lysate at 30 µg

Lane 2:

Western blot - Human IL-6 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-il-6-knockout-a549-cell-line-ab273751'>ab273751</a>)

Lane 3:

IL-6 knockout A549 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 4h) cell lysate at 30 µg

Lane 4:

IL-6 knockout A549 IL-1β (<a href='/en-us/products/proteins-peptides/recombinant-human-il-1-beta-protein-active-ab259387'>ab259387</a>) (20 ng/ml, 24h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5 ug/ml for the last 4h) cell lysate at 30 µg

Predicted band size: 23 kDa

Observed band size: 25 kDa,35 kDa,40 kDa

false

• WB

Lab

Western blot - Anti-IL-6 antibody [EPR21711] (AB233706)

Lanes 1 - 4 : Merged signal (red and green). Green - ab233706 observed at 25 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab233706 was shown to react with IL-6 in wild-type A549 cells in western blot with loss of signal observed in IL-6 knockout cell line ab273751 (knockout cell lysate ab275501). Wild-type and IL-6 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab233706 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-IL-6 antibody [EPR21711] (ab233706) at 1/1000 dilution

Lane 1:

Wild-type A549 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 4h) cell lysate at 30 µg

Lane 2:

Wild-type A549 IL-1β (<a href='/en-us/products/proteins-peptides/recombinant-human-il-1-beta-protein-active-ab259387'>ab259387</a>) (20 ng/ml, 24h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5 ug/ml for the last 4h) cell lysate at 30 µg

Lane 3:

IL-6 knockout A549 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 4h) cell lysate at 30 µg

Lane 4:

IL-6 knockout A549 IL-1β (<a href='/en-us/products/proteins-peptides/recombinant-human-il-1-beta-protein-active-ab259387'>ab259387</a>) (20 ng/ml, 24h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5 ug/ml for the last 4h) cell lysate at 30 µg

false