Anti-STAT3 antibody [E121-21]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-STAT3 antibody [E121-21] (AB32500)

Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labelling STAT3 with Purified ab32500 at 1 : 20 dilution (5 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150081) secondary antibody was used at 1 : 2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-STAT3 antibody [E121-21] (AB32500)

Immunocytochemistry analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling STAT3 with Purified ab32500 at 1 : 50 dilution (2.1 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IP

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Immunoprecipitation - Anti-STAT3 antibody [E121-21] (AB32500)

ab32500 at 1/20 dilution (0.5 μg) immunoprecipitating STAT3 in HeLa whole cell lysate.

Lane 1 (input) : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab32500 & HeLa whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32500 in HeLa whole cell lysate

For western blotting, ab32500 at 1/1000 and VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Blocking and diluting buffer : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-STAT3 antibody [E121-21] (ab32500)

Predicted band size: 88 kDa

Observed band size: 92 kDa

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Exposure time: 1s

• WB

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Western blot - Anti-STAT3 antibody [E121-21] (AB32500)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : STAT3 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : HEK293 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32500 observed at 92 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32500 detected the expected band for STAT3 in wild-type cells along with additional cross-reactive bands. The band was not seen in STAT3 knockout HAP1 cells. Wild-type and STAT3 knockout samples were subjected to SDS-PAGE. ab32500 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-STAT3 antibody [E121-21] (ab32500)

Predicted band size: 88 kDa

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• WB

Supplier Data

Western blot - Anti-STAT3 antibody [E121-21] (AB32500)

All lanes:

Western blot - Anti-STAT3 antibody [E121-21] (ab32500) at 1/1000 dilution

Lane 1:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HaCaT (Human skin keratinocyte) whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 88 kDa

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