Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling IL-1RA with unpurified ab124962 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Overlay histogram showing HeLa cells fixed in 4% PFA and stained with purified ab124962 at a dilution of 1 in 20 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling IL-1RA with purified ab124962 at 1/100. Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

Anti-IL-1RA antibody [EPR6483] (ab124962) at 20 µg (purified) + L6 whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 20 kDa
Observed band size: 20 kDa

Blocking and dilution buffer: 5% NFDM /TBST.

All lanes : Anti-IL-1RA antibody [EPR6483] (ab124962) at 20 µg (purified)

Lane 1 : A431 whole cell lysate
Lane 2 : HEK293 whole cell lysate
Lane 3 : Molt-4 whole cell lysate
Lane 4 : HeLa whole cell lysate
Lane 5 : Raw264.7 whole cell lysate
Lane 6 : NIH/3T3 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: 20 kDa
Observed band size: 20 kDa

Blocking and dilution buffer: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue labelling IL-1RA with purified ab124962 at a dilution of 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue labelling IL-1RA with purified ab124962 at a dilution of 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling IL-1RA with purified ab124962 at a dilution of 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Flow Cytometry analysis of HeLa cells labelling IL-1RA with purified ab124962 at 1/20 (red). Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

ab124962 (purified) at 1/20 immunoprecipitating IL-1RA in NIH/3T3 whole cell lysate.

Lane 1 (input): NIH/3T3 whole cell lysate (10µg)

Lane 2 (+): ab124962 + NIH/3T3 whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab124962 in NIH/3T3 whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes : Anti-IL-1RA antibody [EPR6483] (ab124962) at 1/10000 dilution (unpurified)

Lane 1 : A431 cell lysates
Lane 2 : 293T cell lysates
Lane 3 : MOLT4 cell lysates
Lane 4 : HeLa cell lysates
Lane 5 : RAW264.7 cell lysates
Lane 6 : NIH 3T3 cell lysates
Lane 7 : L6 cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 20 kDa

Overlay histogram showing A431 cells stained with unpurified ab124962 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab124962, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Equilibrium disassociation constant (K_D)
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