All lanes : Anti-IL-6 antibody [EPR20653] (ab214429) 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: 35 kDa (possible non-specific binding)

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.

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.

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 : Anti-IL-6 antibody [EPR20653] (ab214429) 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 and 300 ng/ml Brefeldin A (BFA) for 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: 21-28 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

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