Overview

  • Product nameAnti-Hsp70 antibody [3A3]See all Hsp70 primary antibodies ...
  • Description
    Mouse monoclonal [3A3] to Hsp70
  • Specificityab5439 detects several members of the heat shock protein 70 kDa (Hsp 70) gene family including Hsp 70, Hsc 70, p75, and following heat shock, Hsp 72 from yeast, Drosophila, fish, mouse, avian, amphibian and human samples.
  • Tested applicationsInhibition Assay, ELISA, Gel supershift assays, Flow Cyt, ICC/IF, ICC, IHC-P, WB, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Cow, Human, Pig, Saccharomyces cerevisiae, Fruit fly (Drosophila melanogaster), Fish, Non Human Primates, Plants, Amphibians
    Predicted to work with: Dog, Bird, Cynomolgus Monkey, African Green Monkey, Bos mutus grunniens
  • Immunogen

    Recombinant fragment corresponding to Human Hsp70 aa 504-617 (N terminal).

  • EpitopeEpitope mapping with a panel of Hsp 70 deletion mutants suggests that the epitope recognized is located between amino acids 504-617 of human Hsp 70, a region that has been shown to be involved in stress-induced nucleolar localization.

Properties

  • FormLiquid
  • Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage bufferPreservative: 0.05% Sodium azide
    Constituent: 99% PBS
  • Concentration information loading...
  • PurityAscites
  • Primary antibody notes The Hsp 70 family is a set of highly conserved proteins that are induced by a variety of biological stresses, including heat stress, in every organism in which the proteins have been examined. The human Hsp 70 family members include: Hsp 70, a protein which is strongly inducible in all organisms but which is also constitutively expressed in primate cells; Hsp 72, a 72 kDa protein that is induced exclusively under stress conditions; Hsc 70, or cognate protein, is a 72 kDa, constitutively expressed, protein which is involved in the uncoating of clathrin coated vesicles; GRP78, or BiP, is a glucose regulated 78 kDa protein localized in the endoplasmic reticulum; and p75, or Hsp 75, a 75 kDa protein that is found within the mitochondria.
  • Clonality Monoclonal
  • Clone number3A3
  • IsotypeIgG1
  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab5439 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
Inhibition Assay Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
Gel supershift assays Use at an assay dependent concentration.
Flow Cyt 1/100.
ICC/IF 1/50 - 1/200.
ICC 1/100. Immunocytochemical staining of Hsp 70 in heat shocked HeLa cells with ab5439 results in cytoplasmic staining.
IHC-P 1/100. Antigen retrieval is not essential but may optimise staining.
WB 1/1000 - 1/5000. Predicted molecular weight: 70-78 kDa.

Representing different members of the HSP70 family. 2-dimensional gel electrophoresis is required to resolve the heat induced form of these proteins from their constitutively expressed counterparts.

IP Use a concentration of 2 µg/ml.

Target

  • FunctionIn cooperation with other chaperones, Hsp70s stabilize preexistent proteins against aggregation and mediate the folding of newly translated polypeptides in the cytosol as well as within organelles. These chaperones participate in all these processes through their ability to recognize nonnative conformations of other proteins. They bind extended peptide segments with a net hydrophobic character exposed by polypeptides during translation and membrane translocation, or following stress-induced damage. In case of rotavirus A infection, serves as a post-attachment receptor for the virus to facilitate entry into the cell.
  • Tissue specificityHSPA1B is testis-specific.
  • Sequence similaritiesBelongs to the heat shock protein 70 family.
  • Cellular localizationCytoplasm. Localized in cytoplasmic mRNP granules containing untranslated mRNAs.
  • Information by UniProt
  • Database links
  • Alternative names
    • DAQB 147D11.1 001 antibody
    • FLJ54303 antibody
    • FLJ54370 antibody
    • FLJ54392 antibody
    • FLJ54408 antibody
    • FLJ75127 antibody
    • Heat shock 70 kDa protein 1 antibody
    • Heat shock 70 kDa protein 1/2 antibody
    • Heat shock 70 kDa protein 1A/1B antibody
    • heat shock 70kDa protein 1A antibody
    • Heat shock 70kDa protein 1B antibody
    • Heat shock induced protein antibody
    • heat shock protein 70 antibody
    • HSP70 1 antibody
    • HSP70 2 antibody
    • HSP70-1/HSP70-2 antibody
    • HSP70-1A antibody
    • HSP70.1 antibody
    • HSP70.1/HSP70.2 antibody
    • HSP70I antibody
    • HSP71_HUMAN antibody
    • HSP72 antibody
    • HSPA1 antibody
    • HSPA1A antibody
    • HSPA1B antibody
    • XXbac BCX40G17.3 001 antibody
    see all

Anti-Hsp70 antibody [3A3] images

  • Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) ab5439 shows staining in MCF-7 cells. Heat Shock Protein 70 staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Heat Shock Protein 70 ab5439 at a dilution of 1/100-1/200 over night at 4 oC washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) ab5439shows staining in HeLa cells. Heat Shock Protein 70 staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Heat Shock Protein 70 ab5439 at a dilution of 1/100-1/200 over night at 4oC washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) ab5439 shows staining in NIH-3T3 cells. Heat Shock Protein 70 staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Heat Shock Protein 70 ab5439 at a dilution of 1/100-1/200 over night at 4oC washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • All lanes : Anti-Hsp70 antibody [3A3] (ab5439) at 1/2500 dilution

    Lane 1 : U2OS cell lysate
    Lane 2 : HeLa cell lysate

    Lysates/proteins at 30 µg per lane.

    Secondary
    HRP-conjugated Goat polyclonal to Mouse at 1/2000 dilution

    Performed under reducing conditions.

    Predicted band size : 70-78 kDa
    Observed band size : 70 kDa (why is the actual band size different from the predicted?)


    Exposure time : 2 minutes

    This image is courtesy of an Anonymous abreview.

    See Abreview

  • Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human Breast carcinoma tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/50 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (ab5439) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human Testis tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/200 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (ab5439) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human Tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/20 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (ab5439) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • Overlay histogram showing Jurkat cells stained with ab5439 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab5439, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

  • Immunoprecipitation of Hsp70 was performed on HeLa cells. Antigen:antibody complexes were formed by incubating 500ug whole cell lysate with 2ul of Hsp70 monoclonal antibody (ab5439) overnight on a rocking platform at 4oC. The immune complexes were captured on 50ul Protein Agarose washed extensively and eluted with Buffer. Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel and transferred to a PVDF membraneand blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a Hsp70 monoclonal antibody (ab5439) at a dilution of 1/1000 overnight rotating at 4oC and probed with goat anti-mouse IgG-HRP secondary antibody at a dilution of 1/20,000 for at least 1 hour. Chemiluminescent detection was performed.

References for Anti-Hsp70 antibody [3A3] (ab5439)

This product has been referenced in:
  • Zhao A  et al. Transiently transfected purine biosynthetic enzymes form stress bodies. PLoS One 8:e56203 (2013). ICC/IF ; Human . Read more (PubMed: 23405267) »
  • Maffei S  et al. Beneficial effect of directional freezing on in vitro viability of cryopreserved sheep whole ovaries and ovarian cortical slices. Hum Reprod N/A:N/A (2013). IHC-Fr ; Sheep . Read more (PubMed: 24135077) »

See all 7 Publications for this product

Product Wall

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (8% acrylamide)
Sample Pig Tissue lysate - other (Muscle sarcoplasmic extract)
Specification Muscle sarcoplasmic extract
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Dr. Shannon Cruzen

Verified customer

Submitted Mar 27 2014

Thank you for your enquiry.

I would like to reassure you that both these antibodies are covered by our 6 month guarantee for WB and for human samples.

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