All lanes : Anti-IL-13 receptor alpha 2 antibody [EPR22978-163] (ab260044) at 1/1000 dilution

Lane 1 : Wild-type A375 cell lysate
Lane 2 : IL13RA2 knockout A375 cell lysate
Lane 3 : U-87-MG cell lysate
Lane 4 : Daudi cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Predicted band size: 44 kDa
Observed band size: 49 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab260044 observed at 49 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab260044 was shown to react with IL-13 receptor alpha 2 in wild-type A375 cells in western blot with loss of signal observed in IL13RA2 knockout cell line ab273371 (knockout cell lysate ab275532). Wild-type and IL13RA2 knockout A375 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab260044 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

ab260044 staining IL-13 receptor alpha 2 in wild-type A375 cells (top panel) and IL13RA2 knockout A375 cells (bottom panel) (ab273381). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab260044 at 1/500 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

All lanes : Anti-IL-13 receptor alpha 2 antibody [EPR22978-163] (ab260044) at 1/1000 dilution

Lane 1 : Human testis lysate
Lane 2 : A375 (human malignant melanoma epithelial cell) whole cell lysate
Lane 3 : A-172 (human brain glioblastoma) whole cell lysate
Lane 4 : Daudi (human burkitt's lymphoma lymphoblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/25000 dilution

Predicted band size: 44 kDa
Observed band size: 55,65 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID: 30718742). 

Negative control: Daudi (PMID: 30718742).

Exposure time: Lane 1: 3 minutes; Lane 2: 10 seconds; Lanes 3-4: 3 minutes.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A375 (human malignant melanoma epithelial cell) cells labeling IL-13 receptor alpha 2 with ab260044 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing membranous and cytoplasmic staining in A375 cell line.

Negative control: Daudi (PMID: 26059190.

ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

IL-13 receptor alpha 2 was immunoprecipitated from 0.35 mg A375 (Human malignant melanoma epithelial cell) whole cell lysate 10µg with ab260044 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab260044 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: A375 (Human malignant melanoma epithelial cell) whole cell lysate 10µg

Lane 2: ab260044 IP in A375 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab260044 in A375 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.

 

ELISA analysis of IL13RA2 recombinant protein at 1000 ng/mL with ab260044. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.