All lanes : Anti-IL-6 antibody [EPR21711] (ab233706) at 1/1000 dilution

Lane 1 : Wild-type A549 Brefeldin A (ab120299)-treated (5ug/ml, 4h) cell lysate
Lane 2 : Wild-type A549 IL-1ß (ab259387) (20 ng/ml, 24h) and Brefeldin A (ab120299)-treated (5 ug/ml for the last 4h) cell lysate
Lane 3 : IL-6 knockout A549 Brefeldin A (ab120299)-treated (5ug/ml, 4h) cell lysate
Lane 4 : IL-6 knockout A549 IL-1ß (ab259387) (20 ng/ml, 24h) and Brefeldin A (ab120299)-treated (5 ug/ml for the last 4h) cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Predicted band size: 23 kDa
Observed band size: 25 kDa why is the actual band size different from the predicted?
Additional bands at: 40 kDa (possible non-specific binding)

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.

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 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.

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

Lane 1 : Untreated HUVEC (human umbilical vein endothelial cell line) whole cell lysate
Lane 2 : HUVEC treated with 0.5 µg/ml lipopolysaccharides (LPS) for 24 hours, added 300 ng/ml BFA last 20 hours, whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 23 kDa
Observed band size: 21 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

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

The molecular mass observed is consistent with what has been described in the literature (PMID: 2523818, PMID: 2783321).