Anti-IRS2 antibody [EPR904(2)] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRS2 antibody [EPR904(2)] - BSA and Azide free (AB208195)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134101).

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-IRS2 antibody [EPR904(2)] - BSA and Azide free (AB208195)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134101).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IRS2 antibody [EPR904(2)] - BSA and Azide free (AB208195)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134101).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRS2 antibody [EPR904(2)] - BSA and Azide free (AB208195)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134101).

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)] - BSA and Azide free (AB208195)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134101).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-IRS2 antibody [EPR904(2)] - BSA and Azide free (AB208195)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134101).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRS2 antibody [EPR904(2)] - BSA and Azide free (AB208195)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134101).

• WB

Lab

Western blot - Anti-IRS2 antibody [EPR904(2)] - BSA and Azide free (AB208195)

This data was developed using the same antibody clone in a different buffer formulation (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)] (<a href='/en-us/products/primary-antibodies/irs2-antibody-epr9042-ab134101'>ab134101</a>) 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='/en-us/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

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