Overview

  • Product name
    Anti-Hsp70 antibody [3A3]
    See all Hsp70 primary antibodies
  • Description
    Mouse monoclonal [3A3] to Hsp70
  • Host species
    Mouse
  • Specificity
    ab5439 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 applications
    Suitable for: ELISA, Flow Cyt, ICC/IF, ICC, IHC-P, WB, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Human, Pig, Saccharomyces cerevisiae, Drosophila melanogaster, Fish, Non human primates, Plants, Amphibian
    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).

  • Epitope
    Epitope 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.
  • General notes

     

     

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
  • Concentration information loading...
  • Purity
    Ascites
  • 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 number
    3A3
  • Isotype
    IgG1
  • 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
ELISA Use at an assay dependent concentration.
Flow Cyt 1/100.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ICC/IF 1/50 - 1/200.
EMSA Use at an assay dependent concentration.
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

  • Relevance
    Function: In cooperation with other chaperones, the Hsp70 family 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 specificity: HSPA1B is testis-specific.
  • Cellular localization
    Cytoplasm. Localized in cytoplasmic mRNP granules containing untranslated mRNAs.
  • Database links
  • Alternative names
    • 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 antibody
    • Heat shock 70 kDa protein 1A/1B antibody
    • Heat shock 70kDa protein 1B antibody
    • Heat shock induced protein antibody
    • HEL S 103 antibody
    • HSP70 1 antibody
    • HSP70 1A antibody
    • HSP70 1B antibody
    • HSP70 2 antibody
    • HSP70-1 antibody
    • HSP70-1/HSP70-2 antibody
    • HSP70-1A antibody
    • HSP70.1 antibody
    • HSP70.1/HSP70.2 antibody
    • HSP71_HUMAN antibody
    • HSP72 antibody
    • HSPA1 antibody
    • HSPA1A antibody
    • HSPA1B antibody
    see all

Images

  • All lanes : Anti-Hsp70 antibody [3A3] (ab5439) at 1/2 dilution (12 hours at 4°C)

    Lane 1 : BRAC1 cell lysates in 25% Nupage LSD, 10% DTT, 65% milli with Rockland blocking buffer, 1 hour at 20°C
    Lane 2 : HCT116 cell lysates in 25% Nupage LSD, 10% DTT, 65% milli with Rockland blocking buffer, 1 hour at 20°C
    Lane 3 : K562 cell lysates in 25% Nupage LSD, 10% DTT, 65% milli with Rockland blocking buffer, 1 hour at 20°C
    Lane 4 : SW480 cell lysates in 25% Nupage LSD, 10% DTT, 65% milli with Rockland blocking buffer, 1 hour at 20°C
    Lane 5 : HT29 cell lysates in 25% Nupage LSD, 10% DTT, 65% milli with Rockland blocking buffer, 1 hour at 20°C

    Lysates/proteins at 35 µg per lane.

    Blocking peptides at 100 % per lane.

    Secondary
    All lanes : IRDye 800CW Goat anti-mouse monoclonal at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 70-78 kDa


    Exposure time: 6 minutes

    See Abreview

  • 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.

  • 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.

  • 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.

  • 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.

References

This product has been referenced in:
  • Heymann PGB  et al. Modulation of Tumor Cell Metabolism by Laser Photochemotherapy with Cisplatin or Zoledronic Acid In Vitro. Anticancer Res 38:1291-1301 (2018). Read more (PubMed: 29491052) »
  • Bewicke-Copley F  et al. Extracellular vesicles released following heat stress induce bystander effect in unstressed populations. J Extracell Vesicles 6:1340746 (2017). Read more (PubMed: 28717426) »
See all 19 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (cell lysates in 25% Nupage LSD, 10% DTT, 65% milli)
Gel Running Conditions
Reduced Denaturing (12.5% Acrylamide gel)
Loading amount
35 µg
Specification
cell lysates in 25% Nupage LSD, 10% DTT, 65% milli
Blocking step
Rockland blocking buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 20°C

Abcam user community

Verified customer

Submitted May 27 2015

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

Dr. Shannon Cruzen

Verified customer

Submitted Mar 27 2014

Answer

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.

For more details regarding our guarantee, please see the following page from our website:

https://www.abcam.com/index.html?pageconfig=abpromise

As far as we are aware, the antibodies have not been specifically tried in WB with MCF7 cells. However, this is still likely to work, as they have been tested in WB and human, and so this would still be covered by our guarantee.

Also, you can obtain rewards by submitting an Abreview about these product via the online product datasheets. We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to researchers.

To find out more about our Abreview system and the Abpoints rewards you can obtain, please see the following webpage:

https://www.abcam.com/abreviews

I hope this will be helpful. Thank you for your time and effort.

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Question
Answer

Thank you for attending our webinar on Immunoprecipitation last week. We hope you enjoyed it and it was useful for you. We would also like to thank you for sending your question and apologize that we were not able to answer it in the Q&A session. The determination of the protein concentration before IP is not different from that for other techniques such as Western blot. Therefore, you could perform a Bradford assay, a Lowry assay or a BCA assay. Bovine serum albumin (BSA) is a frequently-used protein standard. Once you have determined the concentration of each sample, you can freeze them at -20°C or -80°C for later use or prepare for immunoprecipitation or for loading onto a gel. For the actual IP experiment, in a tube add 10-500 μg cell lysate plus the recommended amount of antibody. These amounts will be chosen depending on the abundance of the protein and the affinity of the antibody for the protein, typically in a pilot experiment where a fixed amount of protein is precipitated by increasing amounts of antibody. You can check the antibody datasheet for recommended antibody concentration. As a guideline we recommend: 1 – 5 μl polyclonal antiserum 1 μg affinity-purified polyclonal antibody 0.2 to 1 μl ascites fluid (monoclonal antibody) 20 to 100 μl culture supernatant (monoclonal antibody) I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your message and for confirming these details. I am very pleased to hear the customer would like to accept our offer and test these antibodies in hamster. ab41684 Discount code: #### ab38975 Discount code: #### ab6314 Discount code: #### ab77179 Discount code: #### Expirey date for all these codes: 7th March 2012 This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit the Abreviews for hamster samples and include the relevant code in the “Additional Comments” section so we know the Abreview is for this promotion. 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. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount. If you have any further questions, please do not hesitate to contact us. We look forward to receiving the Abreviews and wish the customer luck with their research.

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Answer

Thank you for your message which has been forwarded to me as Karin is currently away from the office. I have reviewed these three antibodies with respect to discount codes: ab77179 I am sorry I am also unable to check the alignment of the immunogen with the hamster sequence, because there is regrettably no hamster sequence available on the protein database sites. I would be pleased to make an exception and provide a discount code in this case anyway, to test in Hamster. ab6314 There is 73% Alignment of the human full length immunogen to the hamster sequence you have provided. This is a little low, but there may be some crossreactivity so I would be pleased to provide a discount. ab16045 There is 14% alignment of the hamster sequence provided with the immunogen. This is very low so the antibody will probably not detect in hamster, and so I am sorry we would be unable to provide a discount for this particular antibody to test in hamster. I hope this will be helpful to you. Please confirm if the customer would like to receive a code for testing ab6314 and ab77179 in hamster and I will be pleased to arrange this for you.

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Answer

Thank you for contacting us. If an antibody has not been tested with a particular species then a testing discount would apply. However, in order to check the likelihood of the antibody being reactive against the target the hamster protein sequence needs to be compared to that of the immunogen used to make sure that the most suitable antibody is chosen. Unfortunately I have been unable to find sequences for Entactin, Cathepsin L or Cyclophilin B for hamster so if you or your customer could help me with this that would be very useful. We already have several antibodies directed against Hsp70 which have been tested with hamster samples. ab47454 is a mouse monoclonal or ab79852 is a rabbit polyclonal. We also have several which are directly conjugated that may be of interest too. These can be found from the following link: https://www.abcam.com/index.html?pageconfig=searchresults&search=hsp70&pt=1&sk=app&sv=69&sn=WB&l=3&fViewMore=1 The Cyclophilin A antibody suggested (ab41684) is predicted to react and would therefore be suitable for a testing discount. However, with the TIMP2 antibody suggested (ab109708), the immunogen recognises the N-terminal sequence of the protein which differs significantly from that of the hamster protein. I would therefore recommend ab38975 as an alternative. This is a rabbit polyclonal directed against the C-terminal end which shares significant homology with Chinese hamster sequence (SwissProt Q60453) compared to the human (SwissProt P16035). I hope this information is helpful to you. If you'd like me to issue testing discounts for ab41684 or ab38975. Or you would like to stick with the original Hsp70 antibody recommended please let me know. Please do not hesitate to contact us if you need any more advice or information.

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Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing (12% Tris Glycine)
Sample
Human Cell lysate - whole cell (U2OS and HeLa cells)
Specification
U2OS and HeLa cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jan 18 2011

Answer

Thank you for your email. We do not routinely offer free or trial sized samples for testing purposes. Our policy at Abcam is that if an antibody does not work as specified on the datasheet, we will offer a replacement or reimbursement. Ab5439 reacts with Amphibians, Chicken, Drosophilia melanogaster, Fish, Human, S. cerevisiae, Mouse and Rat. Therefore, we will guarantee that the antibody will work with Xenopus samples in the following applications that ab5439 has been tested in: Western blot, Immunoprecipitation, Immunohistochemistry (Formalin-fixed paraffin-embedded sections), and Immunocytochemistry. Please note that ab5439 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. As far as we are aware, cross reactivity with Xenopus has not yet been tested for use with ab5444 (so far it has only been tested for corss reactivity with human). Should you decide to go ahead and purchase this product, please let us know how you get on by submitting an Abreview and in return we will award you 50 Abcam Points, which can be redeemed on a number of rewards (a further 100 points will be offered for an image). If you have any additional questions, please contact us again.

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