Anti-IL-18 antibody [EPR19954]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IL-18 antibody [EPR19954] (AB207324)

Flow cytometry overlay histogram showing left PC3 positive cells and right negative Jurkat stained with ab207324 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 mins. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab207324) (1x 10⁶ in 100μl at 1μg/ml (1/2,500 dilution)) for 30 mins at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/2000 dilution for 30 mins at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-18 antibody [EPR19954] (AB207324)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed PC-3 (Human prostate adenocarcinoma cell line) cells labeling IL-18with 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 Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-18 antibody [EPR19954] (AB207324)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HaCaT (Human keratinocyte cell line) cells labeling IL-18with 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 Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-18 antibody [EPR19954] (AB207324)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed Jurkat (Human T cell leukemia cell line from peripheral blood) cells labeling IL-18with 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 Fluorr^® 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.

• WB

Supplier Data

Western blot - Anti-IL-18 antibody [EPR19954] (AB207324)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-IL-18 antibody [EPR19954] (ab207324) at 1/1000 dilution

Lane 1:

PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate at 10 µg

Lane 2:

HaCaT (Human keratinocyte cell line) whole cell lysate at 10 µg

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 4:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-IL-18 antibody [EPR19954] (AB207324)

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:

Western blot - Anti-IL-18 antibody [EPR19954] (ab207324) at 1/200 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

IL18 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human IL18 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-il18-knockout-hela-cell-line-ab265274'>ab265274</a>)

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

false

• WB

Lab

Western blot - Anti-IL-18 antibody [EPR19954] (AB207324)

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.

All lanes:

Western blot - Anti-IL-18 antibody [EPR19954] (ab207324) at 1/200 dilution

Lane 1:

Wild-type HEK-293 whole cell lysate at 20 µg

Lane 2:

IL-18 knockout HEK-293 whole cell lysate at 20 µg

Predicted band size: 22 kDa

false

• WB

Supplier Data

Western blot - Anti-IL-18 antibody [EPR19954] (AB207324)

Blocking/Dilution buffer : 5% NFDM/TBST.

Detection in skin and ovary required high concentration of antibody.

All lanes:

Western blot - Anti-IL-18 antibody [EPR19954] (ab207324) at 1/200 dilution

Lane 1:

Human skin lysate at 10 µg

Lane 2:

Human ovary cancer lysate at 10 µg

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

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-IL-18 antibody [EPR19954] (AB207324)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-IL-18 antibody [EPR19954] (ab207324) at 1/1000 dilution

All lanes:

Human IL-18 active protein at 0.02 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 22 kDa

Observed band size: 17 kDa

false

Exposure time: 3min