Overview

  • Product nameAnti-Hsp70 [2A4] antibodySee all Hsp70 primary antibodies ...
  • Description
    Mouse monoclonal [2A4] to Hsp70
  • Specificityab5442 detects several members of the heat shock protein 70 kDa (Hsp 70) gene family including Hsp 70, Hsc 70 and, following heat shock, Hsp 72 from yeast, Drosophila, fish, mouse, avian, amphibian and human samples. Immunofluorescence staining of Hsp 70 in heat shocked HeLa cells with ab5442 results in cytoplasmic staining.
  • Tested applicationsFlow Cyt, Inhibition Assay, ICC, IP, ICC/IF, IHC-P, WB more details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Human, Saccharomyces cerevisiae, Fruit fly (Drosophila melanogaster), Fish, Non Human Primates, Amphibians
    Predicted to work with: Cow, Pig
  • Immunogen

    Recombinant fragment corresponding to Human Hsp70.

  • EpitopeEpitope mapping with a panel of Hsp 70 deletion mutants suggests that the epitope recognized is located between amino acids 437-479 of human Hsp 70.
  • Positive control
    • ICC: heat shocked HeLa cells

Properties

  • FormLiquid
  • Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage bufferPreservative: 0.05% Sodium azide
    Constituent: 99% PBS
  • Concentration information loading...
  • PurityProtein A purified
  • 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 number2A4
  • IsotypeIgM
  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab5442 in the following tested applications.

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

Application Notes
Flow Cyt Use 1µg for 106 cells.
Inhibition Assay Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
IP Use a concentration of 2 µg/ml.
ICC/IF 1/100 - 1/200.
IHC-P 1/200.

Antigen retrieval is not essential but may optimise staining (using a heat mediated method with citrate buffer).

WB 1/1000 - 1/2500.

Detects a band of approximately 70-72 kDa representing different members of the Hsp 70 family. 2-dimensional gel electrophoresis is required to resolve the heat induced form of these proteins from their constitutively expressed counterparts.

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.
  • Target information above from: UniProt accession P08107 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    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 [2A4] antibody images

  • Immunocytochemistry/Immunofluorescence analysis of Hsp70 (green) in Hela cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with ab5442 at a dilution of 1:100 and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI. Images were taken at a 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of Hsp70 (green) in A431 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with ab5442 at a dilution of 1:100 and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI. Images were taken at a 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of Hsp70 (green) in NIH-3T3 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with ab5442 at a dilution of 1:200 and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI. Images were taken at a 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of Hsp70 in HeLa Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were incubated without (control) or with ab5442 at a dilution of 1:200 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Hsp70 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of Hsp70 in NCI-H1299 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab5442 at a dilution of 1:100 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Hsp70 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of Hsp70 in NIH-3T3 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab5442 at a dilution of 1:100 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Hsp70 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

  • Immunoprecipitation of Hsp70 was performed on HeLa cells. Antigen-antibody complexes were formed by incubating 500ug of whole cell lysate with 2ug of HSP70 monoclonal antibody (ab5442) overnight on a rocking platform at 4�C. The immune complexes were captured on 50ul Protein A/G Agarose and eluted with Buffer. Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membraneand blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a Hsp70 monoclonal antibody (ab5442) at a dilution of 1:1000 overnight rotating at 4�C then washed in TBST and probed with a goat anti-mouse IgM secondary antibody at a dilution of 1:20000 for at least 1 hour. Chemiluminescent detection was performed.
  • Immunocytochemistry/Immunofluorescence analysis of Hsp70 (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% Blocker BSA for 15 minutes at room temperature. Cells were probed with ab5442 at a dilution of 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with fluorescently labeled goat anti-mouse IgM secondary antibody at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.

  • Ab5442 staining human normal skin. Staining is localised to the cytoplasm and nucleus.
    Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
    Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required
  • Immunohistochemistry was performed on normal 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 1:200 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 ab5442 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 cancer biopsies of deparaffinized Human prostate 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 1:200 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 ab5442 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 normal biopsies of deparaffinized Human breast 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 ab5442 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 ab5442 (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 (ab5442, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgM (mu chain) (ab97007) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgM [ICIGM] (ab91545, 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.

References for Anti-Hsp70 [2A4] antibody (ab5442)

This product has been referenced in:
  • Tremosini S  et al. Prospective validation of an immunohistochemical panel (glypican 3, heat shock protein 70 and glutamine synthetase) in liver biopsies for diagnosis of very early hepatocellular carcinoma. Gut 61:1481-7 (2012). ICC/IF ; Human . Read more (PubMed: 22287594) »
  • Zampell JC  et al. Temporal and spatial patterns of endogenous danger signal expression after wound healing and in response to lymphedema. Am J Physiol Cell Physiol 300:C1107-21 (2011). WB, IHC-P ; Mouse . Read more (PubMed: 21248077) »

See all 7 Publications for this product

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Thank you for contacting Abcam.

I am sorry about the issues you have been having with ab5442 in IHC-P. As we discussed today, I am sending 2 new vials of ab5442 and one vial of ab45133, all free of charge.

If there is anything else ...

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Thank you for your response. As my colleague has mentioned in her previous e-mail, the PI for this antibody has not been determined since it is not part of the standard QC process. The approximate molecular weight for this antibody is ~900 kDa....

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Application Western blot
Sample Rat Tissue lysate - whole (whole skin)
Loading amount 15 µg
Specification whole skin
Blocking step Milk as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 5%
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Submitted Jan 31 2007

Thank you for contacting us on behalf of your customer regarding a problem with ab5442. The customer's protocol is very good and I have one small question to finish my assessment of the problem: what dilutions has he tried? I confirmed with the lab...

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