All lanes : Anti-IL-18 antibody [EPR19954] (ab207324) at 1/200 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : IL18 knockout HeLa cell lysate
Lane 3 : A431 cell lysate
Lane 4 : Jurkat cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 22 kDa
Observed band size: 22 kDa

Lanes 1-4: Merged signal (red and green). Green - ab207324 observed at 22 kDa. Red - loading control ab8245 observed at 37 kDa.  

 ab207324 Anti-IL-18 antibody [EPR19954] was shown to specifically react with IL-18 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265274 (knockout cell lysate ab256952) was used. Wild-type and IL-18 knockout samples were subjected to SDS-PAGE. ab207324 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) were incubated overnight at 4°C at 1 in 200 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-IL-18 antibody [EPR19954] (ab207324) at 1/200 dilution

Lane 1 : Wild-type HEK-293 whole cell lysate
Lane 2 : IL-18 knockout HEK-293 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 22 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab207324 observed at 22 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab207324 was shown to recognize IL-18 in wild-type HEK293 cells as signal was lost at the expected MW in IL-18 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and IL-18 knockout samples were subjected to SDS-PAGE. Ab207324 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/200 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

Anti-IL-18 antibody [EPR19954] (ab207324) at 1/1000 dilution + Human IL-18 active protein at 0.02 µg

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

Predicted band size: 22 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-IL-18 antibody [EPR19954] (ab207324) at 1/1000 dilution

Lane 1 : PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate
Lane 2 : HaCaT (Human keratinocyte cell line) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The WB profiles are consistent with the literature (PMID 12918059; PMID 11470273).

All lanes : Anti-IL-18 antibody [EPR19954] (ab207324) at 1/200 dilution

Lane 1 : Human skin lysate
Lane 2 : Human ovary cancer lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/5000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Detection in skin and ovary required high concentration of antibody.

Flow cytometric analysis of 4% paraformaldehyde-fixed PC-3 (Human prostate adenocarcinoma cell line) cells labeling IL-18 with ab207324 at 1/60 dilution (red) compared with Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat Anti-Rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde-fixed HaCaT (Human keratinocyte cell line) cells labeling IL-18 with ab207324 at 1/60 dilution (red) compared with Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat Anti-Rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde-fixed Jurkat (Human T cell leukemia cell line from peripheral blood) cells labeling IL-18 with ab207324 at 1/60 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Negative control:

Jurkat cells serve as a negative cell line as described in PMID 15086390.