Overview

  • Product name

    Anti-HSF1 antibody [EP1710Y]
    See all HSF1 primary antibodies
  • Description

    Rabbit monoclonal [EP1710Y] to HSF1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ChIP, WB, IP, Flow Cyt, IHC-P, ICC/IFmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human HSF1 aa 450 to the C-terminus (C terminal). The exact sequence is proprietary.

  • Positive control

    • WB: K562 HAP1 and HeLa whole cell lysate (ab150035). ICC/IF: MCF-7 cells. Flow Cyt: HeLa cells. IHC-P: Human ovarian carcinoma tissue.
  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab52757 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ChIP Use at an assay dependent concentration.
WB 1/50000. Detects a band of approximately 85 kDa (predicted molecular weight: 57 kDa).

For unpurified use at 1/100000. 

IP 1/20.

For unpurified use at 1/100.

Flow Cyt 1/20.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IHC-P 1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF 1/100 - 1/250.

Target

  • Function

    DNA-binding protein that specifically binds heat shock promoter elements (HSE) and activates transcription. In higher eukaryotes, HSF is unable to bind to the HSE unless the cells are heat shocked.
  • Sequence similarities

    Belongs to the HSF family.
  • Domain

    the 9aaTAD motif is a transactivation domain present in a large number of yeast and animal transcription factors.
  • Post-translational
    modifications

    Phosphorylated on multiple serine residues, a subset of which are involved in stress-related regulation of transcription activation. Constitutive phosphorylation represses transcriptional activity at normal temperatures. Levels increase on specific residues heat-shock and enhance HSF1 transactivation activity. Phosphorylation on Ser-307 derepresses activation on heat-stress and in combination with Ser-303 phosphorylation appears to be involved in recovery after heat-stress. Phosphorylated on Ser-230 by CAMK2, in vitro. Cadmium also enhances phosphorylation at this site. Phosphorylation on Ser-303 is a prerequisite for HSF1 sumoylation. Phosphorylation on Ser-121 inhibits transactivation and promotes HSP90 binding. Phosphorylation on Thr-142 also mediates transcriptional activity induced by heat. Phosphorylation on Ser-326 plays an important role in heat activation of HSF1 transcriptional activity.
    Sumoylated with SUMO1 and SUMO2 on heat-shock. Heat-inducible sumoylation occurs after 15 min of heat-shock, after which levels decrease and at 4 hours, levels return to control levels. Sumoylation has no effect on HSE binding nor on transcriptional activity. Phosphorylation on Ser-303 is a prerequisite for sumoylation.
  • Cellular localization

    Cytoplasm. Nucleus. Cytoplasmic during normal growth. On activation, translocates to nuclear stress granules. Colocalizes with SUMO1 in nuclear stress granules.
  • Information by UniProt
  • Database links

  • Alternative names

    • Heat shock factor 1 antibody
    • Heat shock factor protein 1 antibody
    • Heat shock transcription factor 1 antibody
    • HSF 1 antibody
    • hsf1 antibody
    • HSF1_HUMAN antibody
    • HSTF 1 antibody
    • HSTF1 antibody
    see all

Images

  • All lanes : Anti-HSF1 antibody [EP1710Y] (ab52757) at 1/100000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : Hsf1 knockout HAP1 whole cell lysate
    Lane 3 : Hela whole cell lysate
    Lane 4 : K562 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 57 kDa
    Observed band size: 57 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab52757 observed at 57 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab52757 was shown to specifically react with in wild-type HAP1 cells as signal was lost in Hsf1 knockout cells. Wild-type and Hsf1 knockout samples were subjected to SDS-PAGE. Ab52757 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/100000 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.

  • Anti-HSF1 antibody [EP1710Y] (ab52757) at 1/50000 dilution (purified) + HeLa (Human cervix adenocarcinoma epithelial cell ) whole cell lysates at 15 µg

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

    Predicted band size: 57 kDa
    Observed band size: 80 kDa
    why is the actual band size different from the predicted?



    Blocking and diluting buffer: 5% NFDM/TBST

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human ovarian carcinoma tissue sections labeling HSF1 with Purified ab52757 at 1:250 dilution (1.06 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

  • Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling HSF1 with Purified ab52757 at 1:100 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200. ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

  • ab52757 (purified) at 1:20 dilution (2μg) immunoprecipitating HSF1 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.

    Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
    Lane 2 (+): ab52757 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab52727 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

    For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
    Blocking and diluting buffer: 5% NFDM/TBST.

  • Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling HSF1 with purified ab52757 at 1:20 dilution (10 µg/ml) (red). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

  • Chromatin was prepared from HeLa cells heat shocked (42oC 30 minutes) according to the Abcam Dual X-ChIP protocol. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
    The ChIP was performed with 25 µg of chromatin, 5 µg of ab52757 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (sybr green approach).
    Primers and probes are located in the first kb of the transcribed region.
    *http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

  • Anti-HSF1 antibody [EP1710Y] (ab52757) at 1/100000 dilution (unpurified) + HeLa cell lysate at 10 µg

    Secondary
    HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

    Predicted band size: 57 kDa
    Observed band size: 85 kDa why is the actual band size different from the predicted?

  • Immunocytochemistry/Immunofluorescence analysis of MCF-7 cells labelling HSF1 with unpurified ab52757 at 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.
    Control: PBS only.
    Nuclear counter stain: DAPI.

  • Overlay histogram showing HeLa cells stained with unpurified ab52757 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab52757, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

  • Immunohistochemical staining of paraffin-embedded human ovarian carcinoma using unpurified ab52757 at a 1:100 dilution.

References

This product has been referenced in:

  • Wood RJ  et al. A biosensor-based framework to measure latent proteostasis capacity. Nat Commun 9:287 (2018). Read more (PubMed: 29348634) »
  • Li J  et al. Heat Shock Factor 1 Epigenetically Stimulates Glutaminase-1-Dependent mTOR Activation to Promote Colorectal Carcinogenesis. Mol Ther 26:1828-1839 (2018). Read more (PubMed: 29730197) »
See all 15 Publications for this product

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