Anti-IL-6 antibody [EPR20653]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IL-6 antibody [EPR20653] (AB214429)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HUVEC (human umbilical vein endothelial cell line) cells, untreated or treated with Lipopolysaccharides (LPS) (0.5 μg/ml 24 hours) and Brefeldin A (BFA) (300 ng/ml 20 hours), labeling IL6 with ab214429 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing the cytoplasmic expression increased on HUVEC cells treated with Lipopolysaccharides (LPS) (0.5 μg/ml, 24 hours) and Brefeldin A (BFA) (300 ng/ml, 20 hours).

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

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.

• IP

Supplier Data

Immunoprecipitation - Anti-IL-6 antibody [EPR20653] (AB214429)

IL6 was immunoprecipitated from 0.35 mg of HUVEC (human umbilical vein endothelial cell line) treated with Lipopolysaccharides (LPS) and Brefeldin A (BFA) whole cell lysate with ab214429 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab214429 at 1/1,000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

Lane 1 : HUVEC treated with 0.5 μg/ml LPS for 24 hours and 300 ng/ml BFA for 20 hours whole cell lysate 10 μg (Input).

Lane 2 : ab214429 IP in HUVEC treated with 0.5 μg/ml LPS for 24 hours and 300 ng/ml BFA for 20 hours whole cell lysate (+).

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab214429 in HUVEC treated with 0.5 μg/ml LPS for 24 hours and 300 ng/ml BFA for 20 hours whole cell lysate (-).

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-IL-6 antibody [EPR20653] (ab214429)

Predicted band size: 23 kDa

Observed band size: 21-28 kDa

false

• WB

Supplier Data

Western blot - Anti-IL-6 antibody [EPR20653] (AB214429)

Blocking/Dilution buffer : 5% NFDM/TBST.

The MW observed is consistent with the literature (PMID : 2523818, PMID : 2783321).

All lanes:

Western blot - Anti-IL-6 antibody [EPR20653] (ab214429) at 1/1000 dilution

Lane 1:

Untreated HUVEC (human umbilical vein endothelial cell line) whole cell lysate at 10 µg

Lane 2:

HUVEC treated with 0.5 μg/ml Lipopolysaccharides (LPS) for 24 hours and 300 ng/ml Brefeldin A (BFA) for 20 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: 23 kDa

Observed band size: 21-28 kDa

true

Exposure time: 30s

• WB

Lab

Western blot - Anti-IL-6 antibody [EPR20653] (AB214429)

Lanes 1 - 4 : Merged signal (red and green). Green - ab214429 observed at 25 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab214429 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 ab214429 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 [EPR20653] (ab214429) 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

false

• WB

Lab

Western blot - Anti-IL-6 antibody [EPR20653] (AB214429)

Lanes 1 - 4 : Merged signal (red and green). Green - ab214429 observed at 25 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab214429 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 A549 and IL-6 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab214429 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 [EPR20653] (ab214429) 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