Anti-IL-6 antibody [EPR20653] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IL-6 antibody [EPR20653] - BSA and Azide free (AB245770)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol and sodium azide (ab214429).

• IP

Supplier Data

Immunoprecipitation - Anti-IL-6 antibody [EPR20653] - BSA and Azide free (AB245770)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS#44; BSA#44; glycerol and sodium azide (ab214429)

All lanes:

Immunoprecipitation - Anti-IL-6 antibody [EPR20653] (<a href='/en-us/products/primary-antibodies/il-6-antibody-epr20653-ab214429'>ab214429</a>)

Predicted band size: 23 kDa

Observed band size: 21-28 kDa

false

• WB

Lab

Western blot - Anti-IL-6 antibody [EPR20653] - BSA and Azide free (AB245770)

This data was developed using the same antibody clone in a different buffer formulation (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 [EPR21711] (<a href='/en-us/products/primary-antibodies/il-6-antibody-epr21711-ab233706'>ab233706</a>) 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