Overview

  • Product name
  • Description
    Rabbit polyclonal to HSF1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: EMSA, IP, WB, ELISA, ICC/IF, Inhibition Assaymore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Other Immunogen Type corresponding to Human HSF1. Recombinant human HSF1 expressed in E. coli.

  • Positive control
    • 3T3 cell lysate.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Preservative: 0.05% Sodium azide
    Constituent: 99% PBS
  • Purity
    Whole antiserum
  • Primary antibody notes
    All organisms respond to elevated temperatures and a variety of environmental stresses by rapid synthesis of heat shock RNAs and proteins. The regulation of heat shock gene transcription is mediated by the transcriptional activator, heat shock factor (HSF), which binds to heat shock response elements (HSEs). These HSEs are found as three repeats of a 5-nucleotide {nGAAn} module, arranged in alternating orientation and present upstream of all heat shock genes. The HSEs are highly conserved among species yet HSF purified from yeast, Drosophila and human have different molecular weights and the proteins do not show significant immunological cross reaction. Two HSFs have been identified in human cells, HSF 1 and HSF 2, which bind to the same HSEs and have 38% sequence identity. These factors are activated by distinct stimuli, HSF 1 is responsive to classical stress signals such as heat, heavy metals and oxidative reagents, whereas HSF 2 is activated during hemin-mediated differentiation of human erythroleukemia cells. HSF 1 exists constitutively in the cytoplasm and the nucleus of unstressed cells as a monomer which lacks DNA binding activity. Through an unknown signal generated during stress, HSF 1 becomes activated to a nuclear localized, trimeric state which binds to DNA. The phosphorylation of HSF 1 is necessary for maximal transcription of heat shock genes.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab2923 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
EMSA Use at an assay dependent concentration. PubMed: 18429957
EMSA Use at an assay dependent concentration.
IP Use at an assay dependent concentration.

2 μg

WB 1/1000 - 1/10000. Detects a band of approximately 83 kDa (predicted molecular weight: 57 kDa).
ELISA Use at an assay dependent concentration.
ICC/IF 1/50.
Inhibition Assay 1/1000.

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

  • Western blot analysis of Heat Shock Factor 1 (HSF1) was performed by loading 50µg of the indicated whole cell lysates per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was incubated with ab2923 (1/1000) overnight at 4°C on a rocking platform, washed in TBS-0.1% Tween 20, and probed with a HRP-conjugated goat anti-rabbit IgG secondary antibody (1/20,000) for at least one hour. Chemiluminescent detection was performed.

  • Immunocytochemistry/Immunofluorescence analysis of HSF1 (green) in HeLa and NIH3T3 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% BSA for 15 minutes at room temperature. Cells were probed with ab2923 (1:50) for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488-conjugated goat-anti rabbit IgG secondary antibody (1:400) for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.

  • Immunoprecipitation of HSF1 was performed on HeLa cells. Antigen:antibody complexes were formed by incubating 500µg whole cell lysate with 2µg of ab2923 overnight on a rocking platform at 4°C. Immune complexes were captured on 50µl Protein A/G Plus Agarose, washed extensively, and eluted with buffer. Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBST for at least 1 hour. The membrane was incubated with ab2923 (1:1000) overnight rotating at 4°C, washed in TBST, and probed with detection reagent (1:1000) for at least one hour. Chemiluminescent detection was performed.

References

This product has been referenced in:
  • Grossi V  et al. The longevity SNP rs2802292 uncovered: HSF1 activates stress-dependent expression of FOXO3 through an intronic enhancer. Nucleic Acids Res 46:5587-5600 (2018). Read more (PubMed: 29733381) »
  • Neueder A  et al. Novel Isoforms of Heat Shock Transcription Factor 1, HSF1?a and HSF1?ß, Regulate Chaperone Protein Gene Transcription. J Biol Chem 289:19894-906 (2014). Read more (PubMed: 24855652) »
See all 10 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Question
Answer

Thank you for contacting us regaridng testing of these product in C.elegans. I have created two codes for you. Please be aware that each code is linked to a specific antibody. I am very pleased to hear you would like to accept our offer and test these in c.elegans. Each code will give you: 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for c.elegans and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews. Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Thank you for contacting Abcam. I have processed a free of charge replacement for ab2923. If there is anything else I can help with, please let me know.

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Answer

The cells were heat shocked at ~44 degrees celcius at different time points from 0-6 hours. We unfortunately don't have anymore exact information. If you have any additional questions, please contact us again.

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Question

DESCRIPTION OF THE PROBLEM Non-specific bands...there are several non specific bands--all lower molecular weight than HSF1 when lysate of the cell expressing HSF1 is run. In other words, these bands are only seen when expressing FLAG-HSF1 both in Sf9 lysate, and E. Coli lysate. When ran either GST-HSF1 expressing E. Coli lysate or purified HSF1, only see one clean band. No band is detected with lysate from Sf9 cells that were not infected with HSF1 baculovirus (no expression of FLAG-HSF1) as expected. SAMPLE Sf9 whole cell extract, and E. Coli lysate PRIMARY ANTIBODY ab2923 (aHSF1) from abcam. Rabbit polyclonal. Incubated blot in 1:10,000 of primary diluted into blocking buffer overnight at 4C, washed 3x in TBST (0.5% Tween) DETECTION METHOD Immun-Star detection from BioRad (ECL/Luminol based) POSITIVE AND NEGATIVE CONTROLS USED recombinant HSF1 as positive control, lysate of uninfected Sf9 cells ANTIBODY STORAGE CONDITIONS Primary stock was diluted 1:20 in TBS with BSA (%?) and frozen at -20C. To use, the diluted stock is thawed, and further diluted into TBST (0.1% Tween) with 5% milk. Remaining antibody is left at 4C until needed--never refrozen. After use for blotting, the antibody in the milk solution is refrozen at -20 and reused again next time. SAMPLE PREPARATION Protease inhibitors are added as in typical cocktail mixtures. Sf9 cells were dounce homogenized, lysate was spun at 100,000g-ran supernatant. E.Coli cells were resuspended in PBS with 0.3M NaCl, 0.1% Tween 20, sonicated, spun down debris, and then frozen at -70C. Bolied samples at 100C for 5 minutes in typical 4X loading buffer: Tris, SDS, glycerol, B-ME, DTT. AMOUNT OF PROTEIN LOADED not sure.. significant amount. All samples have the same amount of lysate loaded. ELECTROPHORESIS/GEL CONDITIONS 10% SDS-PAGE gel , reducing TRANSFER AND BLOCKING CONDITIONS Transfered at 100V for 45 min. in Tris/Glycine/10% MeOH Blocked for 30 min. at RT in TBST (0.1% Tween) with 5% milk SECONDARY ANTIBODY BioRad Goat anti Rabbit-HRP 1:15,000 or 1:30,000 in blocking buffer for 1 hr at RT, wash 3x in TBST (0.5% Tween) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 15 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? blocking reagent, blocking time, secondary concentrations, wash buffer Tween concentration

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Answer

Thank you for your enquiry. I am sorry to hear that you are having problems with your anti-HSF1 (ab2923) antibody. Following on from your on line submission. It seems that you are obtaining good quality results with your GST-HSF1 fusion positive control but not from your FLAG tagged positive control which seems to be producing bands of smaller molecular weight following western blotting. It seems from what you have submitted that your FLAG-HSF1 construct may be producing truncated products that are being labelled on the western blot because of the presence of the FLAG tag. May I suggest that you move the FLAG tag to the C-terminal end. This way whilst truncated products may be produced, only completed proteins will contain their FLAG tag and therefore be labelled on the western blot. This may be worth thinking about, especially if moving the tag will not affect the proteins interactions in complexes or the activity of an enzyme. Please do not hesitate to contact me in the future should you require further assistance.

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Question

BATCH NUMBER 100409 ORDER NUMBER 1249031 DESCRIPTION OF THE PROBLEM Multiple bands where HSF1 should be observed. SAMPLE Mouse hepatoma (liver) cells Wild type and knockout Heat Shock Factor-1 mouse embroyonic stem cells from Dr. I. Benjamin PRIMARY ANTIBODY Abcam/Rabbit Polyclonal Antibody to HSF1 (ab2923) 1:10,000, 2% Blotto Incubate overnight at 4 degrees Celsius and have tried 1 hour at room temperature. Varied Blotto amount as well SECONDARY ANTIBODY Amersham/ Donkey Anti-Rabbit Labelled Horseradish Peroxidase 1:2000, 2% Blotto Have never had a problem with this antibody. Have conducted control experiment where no primary antibody added and only secondary antibody used. This antibody does not pick up bands unless Rabbit polyclonal antibody used first. DETECTION METHOD Use ECL Plus kit and follow their directions on washes with TBS-Tween. Have tried using Reagent A: 5ml with Reagent B: 125ul and Reagent A: 5ml with Reagent B: 63ul to cut down on signal and background Have never had a problem with this kit. POSITIVE AND NEGATIVE CONTROLS USED HSF1 protein (cDNA construct) added to gel. Secondary antibody used without primary antibody. Wild type and knockout HSF1 mouse embronic stem cells - Data published on these cells lines (I. Benjamin). Used primary antibody from Stressgen (Cat# SPA-950) and this works well on these extracts and blots (after restripped and re-probed with new antibody) ANTIBODY STORAGE CONDITIONS Antibody is stored at 4 degrees Celsius. SAMPLE PREPARATION Whole Cell Buffer: 20mM Hepes (pH 7.9), 1.5mM MgCl2, 410mM KCL, 0.2mM EDTA, 25% glycerol, DTT, protease inhibitors, phosphatase inhibitors, NaF, and sodium orthovanadate, and PMSF. Boil samples 4 minutes, centrifuge 5 minutes at 13,000 rpm, measure concentration. Have also used 1X Laemilli Sample Buffer (SDS) with protease inhibitors, phosphatase inhibitors, NaF, sodium orthovanadate, and PMSF. Boil samples 4 minutes, centrifuge 5 minutes at 13,000 rpm, measure concentration. AMOUNT OF PROTEIN LOADED Twenty and thirty micrograms ELECTROPHORESIS/GEL CONDITIONS Reducing, 4-12% TB-NuPage gels from Invitrogen TRANSFER AND BLOCKING CONDITIONS 1X Transfer Buffer: 25mM Tris-HCl, 192mM Glycine, and 20% Methanol 1 hour and 20 minutes Experiments: 5% Blotto (TBS-tween and non-fat dry milk) Blocked overnight at 4 degrees Celsius or 2 hours at room temperature on rocker. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? I have altered concentration of antibody from 1:1000 to 1:10,000 and have altered the blotto from 2% to 5%. Have also played around with time (overnight at 4 degrees Celsius to 1 hour at room temperature) and temperature (room temperature vs. 4 degrees Celsius) that antibody is exposed to blot. ADDITIONAL NOTES Used primary antibody from Stressgen (Cat# SPA-950) and this works well on these extracts and blots (after restripped and re-probed with new antibody)Would like refund since using the HSF1 antibody (Cat# SPA-950) from Stressgen and works better. Looking at wild type and knock out we are seeing too many bands in knockout HSF1 cells where the HSF1 protein should not be observed.

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Answer

Thank you for your enquiry regarding ab2923. I am sorry to hear that you have had problems with this antibody. You have done alot of work to optimize it and your procedure looks very good. I can send you a replacement vial of a different lot number free of charge, in case the problem is with that lot number, or if you would prefer I can send you a credit note towards a future purchase or a refund. Please let me know what you would prefer and I look forward to hearing from you.

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