All lanes : Anti-IRS2 antibody [EPR904(2)] (ab134101) at 1/1000 dilution

Lane 1 : Wild-type HEK-293 cell lysate
Lane 2 : IRS2 knockout HEK-293 cell lysate
Lane 3 : A-375 cell lysate
Lane 4 : A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 137 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab134101 observed at 160 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab134101 was shown to react with IRS2 in wild-type HEK-293 cells in western blot with loss of signal observed in IRS2 knockout sample. Wild-type and IRS2 knockout HEK-293 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab134101 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.

ab134101 staining IRS2 in wild-type HEK293 cells (top panel) and IRS2 knockout HEK293 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) 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 ab134101 at 1/500 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).

Immunocytochemistry/Immunofluorescence analysis of SH-SY5Y cells labelling IRS2 with purified ab134101 at 1/300. Cells were fixed with 4% paraformaldehyde 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 (1/300) 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).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue labelling IRS2 with purified ab134101 at 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 IRS2 with purified ab134101 at 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 muscle tissue labelling IRS2 with unpurified ab134101 at a dilution of 1/50.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast tissue labelling IRS2 with unpurified ab134101 at a dilution of 1/50.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-IRS2 antibody [EPR904(2)] (ab134101) at 1/1000 dilution

Lane 1 : Rat liver lysates
Lane 2 : Human liver lysates
Lane 3 : A375 (human malignant melanoma epithelial cell) whole cell lysates
Lane 4 : A549 (human lung carcinoma epithelial cell) whole cell lysates
Lane 5 : NCI-H1299 (human lung carcinoma epithelial cell) whole cell lysates
Lane 6 : LADMAC (Mouse bone marrow monocyte macrophage) whole cell lysates
Lane 7 : MDA-MB-231 (human breast adenocarcinoma epithelial cell) whole cell lysates
Lane 8 : MEF (Mouse embryonic fibroblast) whole cell lysates

Lysates/proteins at 20 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 137 kDa
Observed band size: 170-185 kDa why is the actual band size different from the predicted?

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

Exposure time:

Left image: 180 seconds

Right image: 40 seconds

Although some papers support the expression in liver (PMID: 30202052), A549 (PMID: 30988063), NCI-H1299 (PMID: 30988063), LADMAC (PMID: 29115630), MDA-MB-231 (PMID: 29685905) and MEF (PMID: 30679431), ab134101 can’t detect the target band in these samples, even at the dilution of 1:200.

Anti-IRS2 antibody [EPR904(2)] (ab134101) at 1/5000 dilution (purified) + NIH/3T3 whole cell lysate - treated with insulin at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 137 kDa
Observed band size: 170-185 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-IRS2 antibody [EPR904(2)] (ab134101) at 20 µg (purified)

Lane 1 : HEK293 whole cell lysate - untreated
Lane 2 : HEK293 whole cell lysate - treated with insulin
Lane 3 : SH-SY5Y whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 137 kDa
Observed band size: 170-185 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Flow Cytometry analysis of HeLa cells labelling IRS2 with purified ab134101 at 1/120 (red). Cells were fixed with 2% paraformaldehyde. A FITC-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.

Overlay histogram showing HeLa cells stained with unpurified ab134101 (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 ab134101, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 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. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.