Flow cytometry overlay histogram showing wild-type HeLa (green line) and IL1RAP knockout HeLa cells (ab273375) stained with ab256461 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab256461) (1x10⁶ in 100μl at 10 μ/ml) for 30 min at 4°C.

The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 4°C.

Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type HeLa - black line; IL1RAP knockout HeLa - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

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

All lanes : Anti-IL-1RAcP antibody [EPR22678-67] (ab256461) at 1/1000 dilution

Lane 1 : KARPAS-299 (human anaplastic large cell lymphoma) whole cell lysate
Lane 2 : Raji (human burkitt's lymphoma b lymphocyte) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 65 kDa


Exposure time: 3 minutes

The molecular weight observed is consistent with what has been described in the literature (PMID: 30514753).

Negative control: Raji (PMID: 30514753).

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-IL-1RAcP antibody [EPR22678-67] (ab256461) at 1/1000 dilution + HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

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

Predicted band size: 65 kDa


Exposure time: 3 minutes

The molecular weight observed is consistent with what has been described in the literature (PMID: 30514753).

This blot was developed using a higher sensitivity ECL substrate.

Blocking/Dilution buffer: 5% NFDM/TBT.

Flow cytometric analysis of Raji (Human burkitt's lymphoma b lymphocyte, Left panel) and HepG2 (Human hepatocellular carcinoma epithelial cell, Right panel) cells labeling IL-1RAcP with ab256461 at a 1/60 dilution (1μg, red), compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at a 1/2000 dilution was used as the secondary antibody. Negative control: Raji (PMID: 30514753). Gated on viable cells.

IL-1RAcP was immunoprecipitated from 0.35 mg KARPAS-299 (human anaplastic large cell lymphoma) whole cell lysate 10μg with ab256461 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab256461 at a 1/1000 dilution (0.59 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: KARPAS-299 (human anaplastic large cell lymphoma) whole cell lysate 10μg.

Lane 2: ab256461 IP in KARPAS-299 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab256461 in KARPAS-299 whole cell lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 75 secs.

ELISA analysis of Hu IL1RAP recombinant protein at 1000 ng/mL with ab256461. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.