重组Anti-STAT3 (phospho S727)抗体[E121-31]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT3 (phospho S727) antibody [E121-31] (AB32143)

Immunohistochemical staining of paraffin embedded human astrocytoma with purified ab32143 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-STAT3 (phospho S727) antibody [E121-31] (AB32143)

Purified ab32143 staining STAT3 (phospho S727) in A431 cells by Immunocytochemistry/ Immunofluorescence. 4% PFA-fixed, 0.1% Triton X-100 permeabilized A431 (Human epidermoid carcinoma) cells labelled with ab32143 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor 488) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image showing nuclear staining on A431 cell line. The red staining is ab7291 anti-Tubulin (mouse mAb), followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-STAT3 (phospho S727) antibody [E121-31] (AB32143)

Immunocytochemical/Immunofluorescence analysis of untreated, EGF treated and EGF + LP treated A431 cells labelling STAT3 (phospho S727) with ab32143 (left) and STAT3 with ab32500 (right) both at a dilution of 1/500.

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) (1/1000) was used as the secondary antibody (green). DAPI (blue) was used as the nuclear counterstain. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (1/200) was used as a counterstain (red).

The green staining was increased and translocated from the cytoplasm into the nucleus in the EGF (ab9697 100ng/ml, 10min) treated A431 cells when compared with A431 cells without treatment. After LP treatment, the green signal was decreased. For the pan antibody, there was no great difference after EGF (100ng/ml, 10min) or EGF (100ng/ml, 10min) + LP treatment.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT3 (phospho S727) antibody [E121-31] (AB32143)

Immunohistochemical staining of paraffin embedded mouse liver with purified ab32143 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT3 (phospho S727) antibody [E121-31] (AB32143)

Immunohistochemical staining of paraffin embedded rat cerebral cortex with purified ab32143 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• WB

Lab

Western blot - Anti-STAT3 (phospho S727) antibody [E121-31] (AB32143)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

purified

Lane 1:

A431 treated with epidermal growth factor at 20 µg

Lane 2:

untreated A431 cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/50000 dilution

Predicted band size: 88 kDa

Observed band size: 98 kDa

false

• WB

Unknown

Western blot - Anti-STAT3 (phospho S727) antibody [E121-31] (AB32143)

All lanes:

Western blot - Anti-STAT3 (phospho S727) antibody [E121-31] (ab32143) at 1/1000 dilution

Lane 1:

A431 cell lysate

Lane 2:

A431 + EGF cell lysate

Predicted band size: 88 kDa

Observed band size: 98 kDa

false

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-STAT3 (phospho S727) antibody [E121-31] (AB32143)

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 ab32143 [E121-31]. 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.

• WB

Lab

Western blot - Anti-STAT3 (phospho S727) antibody [E121-31] (AB32143)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-STAT3 (phospho S727) antibody [E121-31] (ab32143) at 1/5000 dilution

Lane 1:

C6 treated with epidermal growth factor at 20 µg

Lane 2:

untreated C6 whole cell lysates at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/50000 dilution

Predicted band size: 88 kDa

Observed band size: 98 kDa

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT3 (phospho S727) antibody [E121-31] (AB32143)

IHC-P analysis of brain astrocytoma using unpurified ab32143 at 1/50 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-STAT3 (phospho S727) antibody [E121-31] (AB32143)

ab32143 (purified) at 1/60 dilution (2.594 μg/ml) immunoprecipitating STAT3 in HeLa whole cell lysate.

Lane 1 (input) : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab32143 & HeLa whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32143 in HeLa whole cell lysate

For western blotting, ab32143 at 1/500 and VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/1000 dilution.

Blocking and diluting buffer : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-STAT3 (phospho S727) antibody [E121-31] (ab32143)

Predicted band size: 88 kDa

false

• Dot

Unknown

Dot Blot - Anti-STAT3 (phospho S727) antibody [E121-31] (AB32143)

Dot Blot analysis of Lane 1 : STAT3 (pS727) phospho peptide and Lane 2 : STAT3 non-phospho peptide labeling STAT3 (phospho S727) with ab32143 at 1/1000 dilution (0.009 μg/ml). 5% NFDM /TBST was used as the diluting and blocking buffer and concentration. ab97051, Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as the secondary antibody at 1/100,000 dilution. Exposure time : 10 seconds.