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

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/µL, 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 downloaded here. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

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.

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

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

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.

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.

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

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

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.