Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR23968-52] to STAT3 (phospho Y705) - BSA and Azide free
  • Suitable for: IP, WB, IHC-P, ICC, Flow Cyt, Dot blot, ChIP
  • Knockout validated
  • Reacts with: Human

Overview

  • Product name

    Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free
    See all STAT3 primary antibodies

  • Description

    Rabbit monoclonal [EPR23968-52] to STAT3 (phospho Y705) - BSA and Azide free

  • Host species

    Rabbit

  • Tested Applications & Species

    Application Species
    ChIP
    Human
    Flow Cyt
    Human
    IP
    Human
    WB
    Human
    See all applications and species data

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: HeLa serum starved overnight, treated 30 mins 50ng/ml IFN alpha; HepG2 serum starved overnight, treated 30 mins, 100ng/ml IL-6; Jurkat treated 30 mins, 50ng/ml IFN alpha. IHC-P: Human endometrial carcinoma, tonsil tissue. ICC: HeLa cells treated with IFN-alpha (50 ng/ml) for 30 min. Flow Cyt: Jurkat treated (30 mins) with 50ng/ml IFN alpha. IP: Jurkat treated (30 mins) with 50ng/ml IFN alpha. Dot: STAT3 phospho Y705 peptide (aa700-710) ChIP: HepG2 (starved overnight) treated with IL-6.

  • General notes

    ab280202 is the carrier-free version of ab267373. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with

    ab280202 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Images

  • Western blot - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    Western blot - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    All lanes : Anti-STAT3 (phospho Y705) antibody [EPR23968-52] (ab267373) at 1/1000 dilution

    Lane 1 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell) serum starved overnight, then treated with 50 ng/ml IFN alpha for 30 minutes, whole cell lysate
    Lane 2 : STAT3 knockout HeLa serum starved overnight, then treated with 50 ng/ml IFN alpha for 30 minutes, whole cell lysate
    Lane 3 : Jurkat (human t cell leukemia cell line from peripheral blood) treated with 50 ng/ml IFN alpha for 30 minutes, whole cell lysate
    Lane 4 : HepG2 (human hepatocellar carcinoma epithelial cell) serum starved overnight, then treated with 100 ng/ml IL-6 for 30 minutes, whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/10000 dilution

    Predicted band size: 88 kDa
    Observed band size: 88 kDa



    This data was developed using ab267373, the same antibody clone in a different buffer formulation. 

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

    Lanes 1-4: Merged signal (red and green). Green - ab267373 observed at 88 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.

    Lanes 1-2: ab267373 Anti-STAT3 (phospho Y705) antibody [EPR23968-52] was shown to specifically react with STAT3 in wild-type serum starved and then IFN alpha treated HeLa cells. Loss of signal was observed when serum starved and then IFN alpha treated knockout cell line ab255436 (knockout cell lysate ab263797) was used. Wild-type and STAT3 knockout samples were subjected to SDS-PAGE. ab267373 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1 in 1000 dilution and 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 10000 dilution for 1 hour at room temperature before imaging.

    Lysates loaded onto lanes 3-4 were made freshly and used in WB immediately to minimize protein degradation.

  • Dot Blot - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    Dot Blot - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)

    This data was developed using ab267373, the same antibody clone in a different buffer formulation.

    Dot blot analysis of STAT3 (phospho Y705) using ab267373 at 1/1000 followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1:100,000 dilution.

    Lane 1: STAT3 phospho Y705 peptide (aa700-710)

    Lane 2: Unmodified STAT3 peptide (aa698-710)

    Exposure time: 3 minutes

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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)

    This data was developed using ab267373, the same antibody clone in a different buffer formulation.

    Immunohistochemical analysis of paraffin-embedded human endometrial carcinoma tissue labeling STAT3 (phospho Y705) with ab267373 at 1/500 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human endometrial carcinoma without alkaline phosphatase treatment (image A) is observed. No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab267373 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

    Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  • Immunocytochemistry - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    Immunocytochemistry - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)

    This data was developed using ab267373, the same antibody clone in a different buffer formulation.

    Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 100% Methanol-permeabilized STAT3 KO HeLa cells labelling STAT3 (phospho Y705) with ab267373 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green).

    Confocal image showing increased nuclear staining in parental HeLa cells treated with IFN-alpha (50 ng/ml) for 30 min, and no staining in STAT3 knockout HeLa cells treated with IFN-alpha (50 ng/ml) for 30 min.

    ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

  • Flow Cytometry - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    Flow Cytometry - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)

    This data was developed using ab267373, the same antibody clone in a different buffer formulation.

    Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Jurkat (Human T cell leukemia T lymphocyte) treated with 50 ng/ml IFN-alpha for 30min (Red)/ Untreated control (Green) cells labelling STAT3 (phospho Y705) with ab267373 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

    A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077 at 1/2000 dilution was used as the secondary antibody.

  • Immunoprecipitation - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    Immunoprecipitation - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)

    This data was developed using ab267373, the same antibody clone in a different buffer formulation.

    STAT3 (phospho Y705) was immunoprecipitated from 0.35 mg Jurkat (human t cell leukemia cell line from peripheral blood) treated with 50 ng/ml IFN alpha for 30 minutes, whole cell lysate 10 µg with ab267373 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab267373 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

    Lane 1: Jurkat treated with 50 ng/ml IFN alpha for 30 minutes, whole cell lysate 10 µg

    Lane 2: ab267373 IP in Jurkat treated with 50 ng/ml IFN alpha for 30 minutes whole cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab267373 in Jurkat treated with 50 ng/ml IFN alpha for 30 minutes whole cell lysate

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

    Exposure time: 15 seconds.

  • ChIP - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    ChIP - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)

    This data was developed using ab267373, the same antibody clone in a different buffer formulation.

    Chromatin was prepared from HepG2 (starved overnight) treated with IL-6 cells and HepG2 (starved overnight) non-treated according to the Abcam Dual-X-ChIP protocol*.

    Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.

    The ChIP was performed with 25 µg of chromatin, 5 µg of ab267373 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
    Primers are purchased from competitor.
    *http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

  • Western blot - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    Western blot - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    All lanes : Anti-STAT3 (phospho Y705) antibody [EPR23968-52] (ab267373) at 1/1000 dilution

    Lane 1 : HepG2 (human hepatocellar carcinoma epithelial cell) serum starved overnight, whole cell lysate
    Lane 2 : HepG2 serum starved overnight, then treated with 100 ng/ml IL-6 for 30 minutes, whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 88 kDa
    Observed band size: 88 kDa



    This data was developed using ab267373, the same antibody clone in a different buffer formulation. 

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

    IL-6 treatment induces phosphorylation of STAT3 at Tyr705 (PMID: 28676732).

    Lysates were made freshly and used in WB immediately to minimize protein degradation.

    Exposure time: 26 seconds.

  • Western blot - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    Western blot - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    All lanes : Anti-STAT3 (phospho Y705) antibody [EPR23968-52] (ab267373) at 1/1000 dilution

    Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) serum starved overnight, whole cell lysate
    Lane 2 : HeLa serum starved overnight, then treated with 50 ng/ml IFN alpha for 30 minutes, whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 88 kDa
    Observed band size: 88 kDa



    This data was developed using ab267373, the same antibody clone in a different buffer formulation. 

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

    IFN alpha treatment induces phosphorylation of STAT3 at Tyr705 (PMID: 21957129).

    Lysates were made freshly and used in WB immediately to minimize protein degradation.

    Exposure time: 3 minutes.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT3 (phospho Y705) antibody [EPR23968-52] - BSA and Azide free (ab280202)

    This data was developed using ab267373, the same antibody clone in a different buffer formulation.

    Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling STAT3 (phospho Y705) with ab267373 at 1/500 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human tonsil without alkaline phosphatase treatment (image A) is observed. No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab267373 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

    Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

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