Anti-IL-18 antibody [EPR19954] - BSA and Azide free

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-18 antibody [EPR19954] - BSA and Azide free (AB222926)

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 Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207324).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-18 antibody [EPR19954] - BSA and Azide free (AB222926)

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 Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207324).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-18 antibody [EPR19954] - BSA and Azide free (AB222926)

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 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207324).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IL-18 antibody [EPR19954] - BSA and Azide free (AB222926)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

• WB

Lab

Western blot - Anti-IL-18 antibody [EPR19954] - BSA and Azide free (AB222926)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207324).

All lanes:

Western blot - Anti-IL-18 antibody [EPR19954] (<a href='/en-us/products/primary-antibodies/il-18-antibody-epr19954-ab207324'>ab207324</a>) 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

Lab

Western blot - Anti-IL-18 antibody [EPR19954] - BSA and Azide free (AB222926)

This data was developed using the same antibody clone in a different buffer formulation (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] - BSA and Azide free (ab222926) 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 3:

A431 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Predicted band size: 22 kDa

Observed band size: 22 kDa

false