All lanes : Anti-IL-18 antibody [EPR19954-188] (ab243091) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : IL-18 knockout HeLa cell lysate
Lane 3 : A431 cell lysate
Lane 4 : Jurkat cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution

Performed under reducing conditions.

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

Lanes 1-4: Merged signal (red and green). Green - ab243091 observed at 22 kDa. Red - loading control ab8245 observed at 37 kDa.  

 ab243091 Anti-IL-18 antibody [EPR19954-188] 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. ab243091 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) were incubated overnight at 4°C at 1 in 1000 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.

 

 

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling IL-18 with ab243091 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic and nuclear staining on distal convoluted tubules of human kidney (PMID: 17687255). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling IL-18 with ab243091 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on Kupffer cells in human liver (PMID: 19084941). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling IL-18 with ab243091 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining on subsets of macrophages in tonsil (PMID: 28842466). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized Jurkat (human T cell leukemia cell line from peripheral blood) (left panel) and PC-3 (human prostate adenocarcinoma cell line) (right panel) cells labeling IL-18 with ab243091 at 1/50 dilution (red) compared with a 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 H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control: Jurkat (PMID 15086390).

All lanes : Anti-IL-18 antibody [EPR19954-188] (ab243091) at 1/1000 dilution

Lane 1 : PC-3 (human prostate adenocarcinoma cell line) whole cell lysate
Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : HaCaT (Human keratinocyte cell line) 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: 22 kDa
Observed band size: 14,22 kDa why is the actual band size different from the predicted?

Exposure time :  Lanes 1 & 2: 3 minutes. Lane 3: 26 seconds.

Blocking and dilution buffer: 5% NFDM/TBST. 

The bands observed are consistent with what has been described in the literature. Mature IL-18 protein is detected as a band at 18 kDa, whereas the 14-kDa band represents
the inactive form (PMID: 15326478).