Lane 1: Wild type HAP1 whole cell lysate (40 µg)
Lane 2: IRAK-1 knockout  HAP1 whole cell lysate (40 µg)
Lane 3: HeLa whole cell lysate (40 µg)
Lane 4: HEK293 whole cell lysate (40 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab208009 observed at 85 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab208009 was shown to specifically react with IRAK-1 when IRAK-1 knockout samples were used. Wild-type and IRAK-1 knockout samples were subjected to SDS-PAGE.  Ab208009 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 200 dilution and 1/10000 dilution respectively. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-IRAK-1 antibody [EPR19667] (ab208009) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 4 : A549 (Human lung carcinoma cell line) 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

Predicted band size: 76 kDa
Observed band size: 68,80 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1/3/4: 1 minute; Lane 2: 30 seconds.

There are two splice variants for IRAK-1, IRAK-1b and IRAK-1c,respectively.   The product recognizes full length IRAK-1 (80 kDa) and IRAK-1c (68 kDa)  (PMID: 16690127).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling IRAK-1 with ab208009 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing mainly cytoplasmic staining on HeLa cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:

-ve control 1: ab208009 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary at 1/1000 dilution.

-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.