Anti-IL-1RA antibody [EPR6483]

• IP

Unknown

Immunoprecipitation - Anti-IL-1RA antibody [EPR6483] (AB124962)

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:

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

All lanes:

NIH/3T3 whole cell lysate

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• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-IL-1RA antibody [EPR6483] (ab124962) at 1/10000 dilution

Lane 1:

Wild-type A431 cell lysate at 20 µg

Lane 2:

IL-1RA knockout A431 cell lysate at 20 µg

Lane 2:

Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-il1rn-il1-receptor-antagonist-knockout-a-431-cell-line-ab273379'>ab273379</a>)

Predicted band size: 20 kDa

Observed band size: 19 kDa

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• WB

Lab

Western blot - Anti-IL-1RA antibody [EPR6483] (AB124962)

False colour image of Western blot : Anti-IL-1RA antibody [EPR6483] staining at 1/50000 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 18 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-IL-1RA antibody [EPR6483] (ab124962) at 1/50000 dilution

Lane 1:

Wild-type A431 cell lysate at 20 µg

Lane 2:

IL1RN knockout A431 cell lysate at 20 µg

Lane 2:

Western blot - Human IL1RN (IL1 Receptor Antagonist) knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-il1rn-il1-receptor-antagonist-knockout-a-431-cell-line-ab273379'>ab273379</a>)

Lane 3:

MOLT-4 cell lysate at 20 µg

Lane 4:

Human Kidney cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 18 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-IL-1RA antibody [EPR6483] (AB124962)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.