Recombinant Anti-IL-1RA antibody [EPR6483] (ab124962)

False colour image of Western blot: Anti-IL-1RA antibody [EPR6483] staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab124962 was shown to bind specifically to IL-1RA. A band was observed at 19 kDa in wild-type A431 cell lysates with no signal observed at this size in IL1RN knockout cell line ab273379 (knockout cell lysate ab275530). To generate this image, wild-type and IL1RN knockout A431 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

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.

Blocking and dilution buffer: 5% NFDM /TBST.

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.

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.