Anti-STAT6 抗体 [E265]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-STAT6 antibody [E265] (AB32108)

Confocal image showing cytoplasmic staining in Raji cells.

Immunocytochemistry/immunofluorescence analysis of Raji (human Burkitt's lymphoma B lymphocyte) cells labelling STAT6 with ab32108 at 10 μg/mL. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) was used as a secondary antibody at 2 μg/mL. Cells were counterstained with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) at 1/200 dilution (red). Nuclear DNA was labelled with DAPI (blue).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-STAT6 antibody [E265] (AB32108)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling STAT6 with purified ab32108 at 1/100 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cells without incubation with primary antibody and secondary antibody (Blue).

• IP

Lab

Immunoprecipitation - Anti-STAT6 antibody [E265] (AB32108)

Purified ab32108 at 1/50 dilution (2μg) immunoprecipitating STAT6 in HeLa whole cell lysate.
Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab32108 + HeLa whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32108 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 100 kDa

All lanes:

Immunoprecipitation - Anti-STAT6 antibody [E265] (ab32108)

Predicted band size: 94 kDa

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• WB

Lab

Western blot - Anti-STAT6 antibody [E265] (AB32108)

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

Exposure time : Lane 1. 7 seconds ; Lane 2. 3 seconds

This antibody recognizes three isoforms of STAT6, isoform 1 (110kDa), isoform 3 (81kDa), which is consistent with the Uniprot annotation.

All lanes:

Western blot - Anti-STAT6 antibody [E265] (ab32108) at 1/10000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Lane 2:

Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysate at 15 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: 94 kDa

Observed band size: 110 kDa,81 kDa

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• WB

Lab

Western blot - Anti-STAT6 antibody [E265] (AB32108)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : STAT6 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : Raji whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32108 observed at 94 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab32108 was shown to specifically react with STAT6 in wild-type HAP1 cells as signal was lost in STAT6 knockout cells. Wild-type and STAT6 knockout samples were subjected to SDS-PAGE. ab32108 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-STAT6 antibody [E265] (ab32108)

Predicted band size: 94 kDa

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