重组Anti-IRS2抗体[EPR904(2)]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-IRS2 antibody [EPR904(2)] (AB134101)

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).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-IRS2 antibody [EPR904(2)] (AB134101)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRS2 antibody [EPR904(2)] (AB134101)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRS2 antibody [EPR904(2)] (AB134101)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-IRS2 antibody [EPR904(2)] (AB134101)

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).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IRS2 antibody [EPR904(2)] (AB134101)

Intracellular 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRS2 antibody [EPR904(2)] (AB134101)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRS2 antibody [EPR904(2)] (AB134101)

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.

• WB

Lab

Western blot - Anti-IRS2 antibody [EPR904(2)] (AB134101)

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.

All lanes:

Western blot - Anti-IRS2 antibody [EPR904(2)] (ab134101) at 1/1000 dilution

Lane 1:

Rat liver lysates at 20 µg

Lane 2:

Human liver lysates at 20 µg

Lane 3:

A375 (human malignant melanoma epithelial cell) whole cell lysates at 20 µg

Lane 4:

A549 (human lung carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 5:

NCI-H1299 (human lung carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 6:

LADMAC (Mouse bone marrow monocyte macrophage) whole cell lysates at 20 µg

Lane 7:

MDA-MB-231 (human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 8:

MEF (Mouse embryonic fibroblast) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 137 kDa

Observed band size: 170-185 kDa

true

• WB

Lab

Western blot - Anti-IRS2 antibody [EPR904(2)] (AB134101)

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.

All lanes:

Western blot - Anti-IRS2 antibody [EPR904(2)] (ab134101) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

IRS2 knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

Western blot - Human IRS2 knockout HEK-293 cell line (<a href='/products/cell-lines/human-irs2-knockout-hek-293-cell-line-ab264013'>ab264013</a>)

Lane 3:

A-375 cell lysate at 20 µg

Lane 4:

A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 137 kDa,25 kDa

Observed band size: 160 kDa

false

• WB

Lab

Western blot - Anti-IRS2 antibody [EPR904(2)] (AB134101)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-IRS2 antibody [EPR904(2)] (ab134101) at 20 µg

Lane 1:

HEK293 whole cell lysate - untreated at 20 µg

Lane 2:

HEK293 whole cell lysate - treated with insulin at 20 µg

Lane 3:

SH-SY5Y whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 137 kDa

Observed band size: 170-185 kDa

false

• WB

Lab

Western blot - Anti-IRS2 antibody [EPR904(2)] (AB134101)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-IRS2 antibody [EPR904(2)] (ab134101) at 1/5000 dilution

All lanes:

NIH/3T3 whole cell lysate - treated with insulin at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 137 kDa

Observed band size: 170-185 kDa

false