Overview

  • Product nameAnti-Hsp70 antibody [5A5]
    See all Hsp70 primary antibodies
  • Description
    Mouse monoclonal [5A5] to Hsp70
  • Tested applicationsIHC-P, WB, ICC/IF, Inhibition Assay, IP, Gel supershift assays, Flow Cyt, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Cow, Human, Saccharomyces cerevisiae, Bird, Fruit fly (Drosophila melanogaster), Fish, Amphibians
  • Immunogen

    Recombinant fragment within Human Hsp70 aa 122-264 (N terminal). The exact sequence is proprietary.

  • EpitopeEpitope mapping with a panel of HSP 70 deletion mutants suggests that the epitope recognized is located between amino acids 122-264 of human HSP 70, a region that has been shown to be involved in ATP binding. This is the first monoclonal antibody reported to react with: 1) The ATP binding region of HSP 70. 2) An epitope in the amino terminus of HSP 70.
  • Positive control
    • Heat shocked HeLa, 293T, K562, A431 and U2OS cells
  • General notes

     

     

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: 100% 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; GRP 78, 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 number5A5
  • IsotypeIgG1
  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab2787 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
IHC-P 1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB 1/1000.

Detects proteins from ~70 kDa to ~78 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.

ICC/IF 1/50 - 1/100.
Inhibition Assay Use at an assay dependent concentration.
IP Use at 1-10 µg/mg of lysate. See Balashova et al.
Gel supershift assays Use at an assay dependent concentration.
Flow Cyt 1/100.
IHC-Fr 1/100 - 1/200.

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
    • DnaK type molecular chaperone HSP70 1 antibody
    • Epididymis secretory protein Li 103 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
    • HEL S 103 antibody
    • HSP70 1 antibody
    • HSP70 1B 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 [5A5] images

  • Western blot analysis of Hsp70 was performed by loading 50µg of the indicated whole cell lysates and 15µl of prestained protein ladder 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 ab2787 (1:1000) overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween 20, and incubated with a goat anti-mouse IgG HRP secondary antibody (1:20,000) for at least 1 hour. Chemiluminescent detection was performed.

  • Immunoprecipitation of Hsp70 was performed on HeLa cells. Antigen:antibody complexes were formed by incubating 500µg whole cell lysate with 2µg of ab2787 overnight on a rocking platform at 4°C. The immune complexes were captured on 50µl Protein A/G Agarose, washed extensively, and eluted with buffer. Samples were then 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 ab2787 (1:1000) overnight rotating at 4°C, washed in TBST, and probed with IP detection reagent-HRP at a dilution of 1:1000 for at least one hour. Chemiluminescent detection was performed.

  • Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (5A5) ab2787 shows staining in U251 glioma 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 ab2787 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 (5A5) ab2787 shows 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 ab2787 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 (5A5) ab2787shows staining in C6 glioma 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 (right) or with or an antibody recognizing Heat Shock Protein 70 (ab2787) (left) 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.

  • ab2787 staining Hsp70 in 2089 cells by Immunocytochemistry/ Immunofluorescence.Cells were fixed in methanol for 30 minutes at -20 °C, washed with PBS, and incubated in blocking solution (10% human serum in PBS) for 1 hour at room temperature. Cells were stained with ab2787 diluted in blocking solution for 1 hour at room temperature in humidified chambers. Cells were washed with PBS and then incubated with secondary antibody diluted 1/200 in blocking solution for 1 hour at room temperature in opaque humidified chambers.


  • developed using the ECL technique

    Observed band size : 70 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 90 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 1 hour

    Image courtesy of an Abreview submitted by Katherina Brokordt.

    ab2787 used in Western Blotting at a 1/1000 dilution.Whole tissue lysate prepared from the gill of the gastropod Concholepas concholepas was loaded at 90µg.The positive control used was bovine Heat shock Cognate 70.Secondary used was an HRP-conjugated rabbit anti-mouse at a 1/5000 dilution.

  • ab2787 staining human skin. Staining is localized to the cytoplasm and nucleus.
    Left panel: with primary antibody at 1/100. Right panel: isotype control.
    Sections were stained using an automated system at room temperature. Sections were rehydrated and antigen retrieved. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 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 (ab2787) 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 1/100 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (ab2787) 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 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 (ab2787) 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 ab2787 (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 (ab2787, 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 (5min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

References for Anti-Hsp70 antibody [5A5] (ab2787)

This product has been referenced in:
  • Kriegenburg F  et al. A chaperone-assisted degradation pathway targets kinetochore proteins to ensure genome stability. PLoS Genet 10:e1004140 (2014). WB ; Schizosaccharomyces pombe . Read more (PubMed: 24497846) »
  • Wen W  et al. VER-155008, a small molecule inhibitor of HSP70 with potent anti-cancer activity on lung cancer cell lines. Exp Biol Med (Maywood) 239:638-45 (2014). Read more (PubMed: 24676905) »

See all 18 Publications for this product

Product Wall

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (6% SDS-PAGE)
Sample Human Cell lysate - whole cell (HeLa)
Specification HeLa
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Dr. Armen Petrosyan

Verified customer

Submitted Nov 12 2014

Application Western blot
Loading amount 25 µg
Gel Running Conditions Reduced Denaturing (4-20% gradient)
Sample Rat Tissue lysate - whole (rat brain)
Specification rat brain
Blocking step Milk as blocking agent for 4 hour(s) and 0 minute(s) · Concentration: 6% · Temperature: RT°C
Username

Dr. Fabrice Gankamk

Verified customer

Submitted Oct 10 2014

Thank you for your recent telephone enquiry.

I can confirm that both ab2790 and ab2787 antibodies are sold as ascites fluid. As discussed on the phone, unpurified antibodies, such as those sold as whole antiserum, ascites or tissue culture s...

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Thank you for providing those details.

The sequences provided are only truncations of the proteins and I have therefore not been able to compare them to the immunogens used to raise the antibodies ab2787 and ab13495. I cannot therefore ascert...

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Thank you very much for providing those sequences.

In order to help the customer, could you please provide me the sequence in text format (i.e. in the body of the email, or as a word document). This will allow me to copy and paste the sequenc...

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Thank you for contacting us.

I have been unable to find a sequence for Hsp90 and Hsp70 in bufo bankorensis, I am therefore not able to say if the antibodies are likely to be able to detect the proteins from these samples. Would you be able to...

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Thank you for your enquiry regarding ab2787.
I can confirm that this product is ascitic fluid and not affinity purified antibody. Thus the concentration is not routinely determined.
However, the concentration of the recent batch is 9.3mg/mL. ...

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Thank you for confirming those details.

I have now arranged for the replacement antibody ab2787 to be sent out to you at the address provided. The order number is xxxxx(Purchase order number FOCR xxxxxx). This should be with you tomorrow. If ...

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Thank you for contacting us. Epitope mapping with a panel of HSP 70 deletion mutants suggests that the epitope recognized is located between amino acids 122-264 of human HSP 70, a region that has been shown to be involved in ATP binding. This region ...

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunoprecipitation
Sample Human Cell lysate - whole cell (huh-7 cells expressing DDDDK tagged recombinant HS)
Total protein in input 1e+006 cells
Specification huh-7 cells expressing DDDDK tagged recombinant HS
Immuno-precipitation step Protein A
Username

Abcam user community

Verified customer

Submitted Jul 29 2010

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"