重组Anti-STAT3抗体[EPR787Y]

• WB

Lab

Western blot - Anti-STAT3 antibody [EPR787Y] (AB68153)

Blocking/Diluting buffer : 5% NFDM/TBST.

Rabbit monoclonal [EPR16891] to GAPDH (ab181602) used as loading control.

All lanes:

Western blot - Anti-STAT3 antibody [EPR787Y] (ab68153) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

C2C12 (Mouse myoblast cell line) whole cell lysate at 20 µg

Lane 5:

Human brain lysate at 20 µg

Lane 6:

Mouse brain lysate at 20 µg

Lane 7:

Rat brain lysate at 20 µg

Lane 8:

Rat kidney 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 1/20000 dilution

Predicted band size: 88 kDa

Observed band size: 92 kDa

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Exposure time: 90s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT3 antibody [EPR787Y] (AB68153)

Immunohistochemical analysis of formalin fixed paraffin embedded human pancreas labelling STAT3 with ab68153 at a concentration of 0.1μg/ml.

The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView HRP/DAB detection kit.

Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab68153 anti-STAT3 antibody [EPR787Y] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-STAT3 antibody [EPR787Y] (AB68153)

ab68153 stainingSTAT3in the human cell line HeLa (human cervix adenocarcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/30. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control : Rabbit monoclonal IgG (Black)

Unlabelled control : Cell without incubation with primary antibody and secondary antibody (Blue)

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-STAT3 antibody [EPR787Y] (AB68153)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling STAT3 (green) with purified ab68153 at 1/200. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT3 antibody [EPR787Y] (AB68153)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human brain tissue sections labelling STAT3 with purified ab68153 at 1/200. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT3 antibody [EPR787Y] (AB68153)

Immunohistochemical analysis of formalin fixed paraffin embedded human skeletal muscle labelling STAT3 with ab68153 at a concentration of 0.1µg/ml.

The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView HRP/DAB detection kit.
Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.
ab68153 anti-STAT3 antibody [EPR787Y] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II.
Image inset shows absence of staining in secondary antibody only control.
Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• WB

Lab

Western blot - Anti-STAT3 antibody [EPR787Y] (AB68153)

Lanes 1 - 4 : Merged signal (red and green). Green - ab68153 observed at 92 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab68153 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. ab68153 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/500 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 [EPR787Y] (ab68153) at 1/500 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

STAT3 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

HEK293 whole cell lysate at 20 µg

Predicted band size: 88 kDa

false

• WB

Lab

Western blot - Anti-STAT3 antibody [EPR787Y] (AB68153)

Lanes 1- 2 : Merged signal (red and green). Green - ab68153 observed at 92 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab68153 was shown to react with STAT3 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255436 (knockout cell lysate ab263797) was used. Wild-type HeLa and STAT3 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab68153 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-STAT3 antibody [EPR787Y] (ab68153) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

STAT3 knockout HeLa cell lysate at 20 µg

Predicted band size: 88 kDa

Observed band size: 92 kDa

false

• WB

Lab

Western blot - Anti-STAT3 antibody [EPR787Y] (AB68153)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab68153 overnight at 4°C. Antibody binding was detected using ab205722, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-STAT3 antibody [EPR787Y] (ab68153) at 1/2000 dilution

Lane 1:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Heart (Mouse) Tissue Lysate at 10 µg

Lane 3:

Heart (Rat) Tissue Lysate at 10 µg

Secondary

All lanes:

Western blot - Donkey Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/donkey-rabbit-igg-h-l-hrp-ab205722'>ab205722</a>) at 1/2000 dilution

Predicted band size: 36 kDa

Observed band size: 88 kDa

true

Exposure time: 20min

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-STAT3 antibody [EPR787Y] (AB68153)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HepG2 cells (starved overnight and treated with 100ng/ml IL-6 for 30min) and 5 µg of ab68153 [EPR787Y]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.