All lanes : Anti-IL-6 antibody [EPR22565-204] (ab233551) at 1/1000 dilution

Lane 1 : Untreated HUVEC (human umbilical vein endothelial cell) whole cell lysate
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

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 24 kDa
Observed band size: 21,24 kDa why is the actual band size different from the predicted?

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

Blocking/Dilution buffer: 5% NFDM/TBST.

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 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.

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.