Anti-Hsp70 antibody [C92F3A-5] (ab47455)

Mouse monoclonal Hsp70 antibody [C92F3A-5]. Validated in WB, IP, ELISA, IHC, Flow Cyt, EM, ICC/IF and tested in Rat, Human. Cited in 23 publication(s). Independently reviewed in 3 review(s).

Overview

  • Product name
    Anti-Hsp70 antibody [C92F3A-5]
    See all Hsp70 primary antibodies
  • Description
    Mouse monoclonal [C92F3A-5] to Hsp70
  • Host species
    Mouse
  • Specificity
    Detects a 70kDa protein corresponding to the molecular mass of Hsp70 of SDS PAGE immunoblots. There is no cross-reactivity to Hsc70 (Hsp73).
  • Tested applications
    Suitable for: Electron Microscopy, WB, IP, ELISA, ICC/IF, Flow Cyt, IHC-Fr, IHC-Pmore details
  • Species reactivity
    Reacts with: Rat, Human
    Predicted to work with: Mouse, Cow, Dog, Pig, Caenorhabditis elegans, Macaque monkey, Bos mutus grunniens, Saguinus oedipus, Onchocerca volvulus, Oncorhynchus tshawytscha
  • Immunogen

    Recombinant fragment corresponding to Human Hsp70 aa 436-503.
    Sequence:

    YSDNQPGVLIQVYEGERAMTKDNNLLGRFELSGIPPAPRGVPQIEVTFDI DANGILNVTATDKSTGKANKITI


    Database link: P08107

  • Epitope
    The mapped epitope is in the region of amino acid residues 436-503.
  • Positive control
    • WB: HeLa, A431, A549, HCT116, HEK293, HepG2, HL-60, HUVEC, Jurkat, MCF7, PC3, T98G cell lysates; Rat Brain tumour lysate; Rat bone lysate. IHC-P: Mouse colon cancer tissue; human colon cancer tissue. Flow cyt: HeLa cells.

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
    pH: 7.20
    Preservative: 0.09% Sodium azide
    Constituents: 50% PBS, 50% Glycerol
  • Concentration information loading...
  • Purity
    Protein G purified
  • Clonality
    Monoclonal
  • Clone number
    C92F3A-5
  • Isotype
    IgG1
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab47455 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
Electron Microscopy Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Predicted molecular weight: 70 kDa.
IP Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
Flow Cyt Use 1µg for 106 cells.

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

IHC-Fr Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.

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 [C92F3A-5] (ab47455) at 1 µg/ml

    Lane 1 : Cell lysate prepared from human A431 cell line
    Lane 2 : Cell lysate prepared from human A549 cell line
    Lane 3 : Cell lysate prepared from human HCT116 cells line
    Lane 4 : Cell lysate prepared from human Hela cell line
    Lane 5 : Cell lysate prepared from human HEK293 cell line
    Lane 6 : Cell lysate prepared from human HepG2 cell line
    Lane 7 : Cell lysate prepared from human HL-60 cell line
    Lane 8 : Cell lysate prepared from human HUVEC cell line
    Lane 9 : Cell lysate prepared from human Jurkat cell line
    Lane 10 : Cell lysate prepared from human MCF7 cell line
    Lane 11 : Cell lysate prepared from human PC3 cell line
    Lane 12 : Cell lysate prepared from human T98G cell line
    Lane 13 : Rat brain tissue lysates

    Predicted band size: 70 kDa



    All are cancer cell lines.

  • Western blot of Hela cell heat shock lysate, using HRP conjugated goat anti-mouse secondary antibody for detection. (1µg/mL ab47455 was used to detect in 10µg of heat shocked HeLa cell lysate)

  • Formalin-fixed, paraffin-embedded human colon carcinoma tissue stained for Hsp70 using ab47455 at 1/1000 dilution in immunohistochemical analysis. Counter stained with hematoxylin.

  • All lanes : Anti-Hsp70 antibody [C92F3A-5] (ab47455) at 1/500 dilution

    All lanes : Rat bone (tibia) whole cell lysate

    Lysates/proteins at 50 µg per lane.

    Predicted band size: 70 kDa

    See Abreview

  • ab47455 staining Hsp70 in mouse colon cancer tissue section by Immunohistochemistry (Bouin's fixed paraffin embedded tissue sections). The primary antibody was diluted at 1/100,000. A Fluorophore conjugated goat anti mouse was used as secondary.  An antibody amplifier™ system was used for staining.

  • Overlay histogram showing HeLa cells stained with ab47455 (red line). The cells were fixed with methanol (5 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 (ab47455, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1:500 dilution for 30 min at 22°C. 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 significantly decreased signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

References

This product has been referenced in:
  • Zhu X  et al. Upconversion nanocomposite for programming combination cancer therapy by precise control of microscopic temperature. Nat Commun 9:2176 (2018). Read more (PubMed: 29872036) »
  • Benitez-Amaro A  et al. Identification of new biophysical markers for pathological ventricular remodelling in tachycardia-induced dilated cardiomyopathy. J Cell Mol Med 22:4197-4208 (2018). Read more (PubMed: 29921039) »
See all 23 Publications for this product

Customer reviews and Q&As

11-17 of 17 Abreviews or Q&A

Question
Answer

Thank you for contacting Abcam.

I am sorry about the issues you have been having with a few of our antibodies. These antibodies are covered under our Abpromise for six months and are guaranteed to work in western blot on rat samples .

For ab47455 and ab49919, I do not think there is any protocol advice I can give that would help resolve this issue, therefore I would be happy to either replace or refund the cost of them. Please let me know how you would like to proceed for these 2 and if you do wish to receive replacements, include the catalogue numbers of the products you would like to receive.

As for ab90537, I see that you used a primary antibody concentration of 1/2000, did you try to use any higher dilutions, say 1/500, with overnight incubation at 4C, to see if that helps resolve the issue?

Looking forward to your reply and hope your day is going well.

Read More

Question

Dear xxxx,
Thank you for your recent reply and find our detailed comments below.
Regards,

1. Please provide the order numbers and dates of purchase

Order Reference # xxxxx Invoice # xxxxxxPurchase date August 5, 2011

We are aware that the 6 months warranty has already expired. Anyway we would like to state that we spent almost 5 months with tests to get the AB working, including tests with Drosophila protein using other ABs and with human serum.

2. Particularly as the antibody has worked well with human samples, could you confirm if there was any optimization of drosophila sample preparation? Was a lysis buffer used? I can recommend to use a lysis buffer such as RIPA to provide a suitable protein preparation from the cells in the tissue.

As I wrote in my original mail, we used PBS supplemented with PMSF and an antiprotease-cocktail containing Pepstatin A, Leupeptin, Benzamindine and Sodiummetabisulfite. This buffer is a standard for Drosophila protein extraction, and we yielded approx. 350 mg total protein from 30 flies.

3. I can suggest that perhaps 30 flies may not generate enough protein for detection, although I do not know enough about using Drosophila to make any predictions about this. I can however suggest to load 20 - 30 ug of total protein per lane of the gel to ensure there is enough protein for detection.

Most Drosophila researchers use 50µl protein solution at a concentration of 25-100 ug/ml to load a single ELISA well; we covered the range 5-500 ug/ml, so a suitable amount of protein should have been present. Furthermore, our Drosophila extract works fine with an ELISA targeting Actin. Thus, I really think the AB does not bind to Drosophila hsp70.

This antibody has been tested in ELISA and drosophila separately, but not together. I am sorry we are not aware of anyone having used this antibody for ELISA with Drosophila before. We know that it works well in WB for drosophila, and it has been used for a wider range of mammalian ELISAs before.

That’s interesting. Could you please provide me a reference to the Drosophila WB? The references on your product web site only cover human, rabbit and pig applications.

regards,

Read More
Answer

Sorry for the delay in getting back to you.

Kate is not in the office today but I will try to help as best I can. As you may know, Kate contacted the source of ab47455 in order to obtain the data collected when the antibody was used in Western blotting with Drosophila samples. Unfortunately, as this result traces back to the 1980's when this clone was initially developed and characterised andthe information has unfortunately been lost and the scientists involved have long since retired. I am therefore sorry but I am unable to provide you with this information.

Having looked through your case I can see that under the conditions used, you would expect to be able to detect the Drosophila protein and I am sorry that this has not been the case. I would like to be able to offer you a replacement antibody to try and to this end I am currently looking into the data collected with some of our other mouse monoclonal antibodies against Hsp70 which have been shown to detect the Drosophila protein to see if there is an appropriate alternative I may be able to offer. I will get back to you as soon as I can with this information.

I understand that the antibody was purchased over 6 months ago and would therefore not normally be covered by the Abpromise. However, I can well appreciate your frustration and the time and effort you have put into optimising this antibody. Therefore, if you would not like to try the alternatives I present to you, I can also arrange for a refund or credit note to be organised.

I look forward to hearing how you would like to proceed.

Read More

Question

Dear Madam/Sir,

Last year we purchased your antibodies AB47455 and AB97020 in order to
detect hsp70 in Drosophila samples. After several unsuccessful attempts
to set up an ELISA protocol, we decided to test the antibodies
additionally on human serum and achieved interesting results.

Protocol outline:
Each 30 Drosophila melanogaster were either exposed to 36°C for 60 min
and thereafter transferred to 25.5oC for 60 min before killing (heat
shock group) or taken directly from the breeding incubator (control
group, 25.5oC) and homogenized in 200 ul PBS/PMSF buffer supplemented
with 1% antiprotease cocktail. The samples were snap frozen in nitrogen
and kept at -70oC. Human serum was collected from one individual by
venipuncture and BD Vacutainer 5ml serum tubes before (control) and 60
min after exposing the individual to 2x20 min 90oC, aka Sauna (heat
shock). Vacutainers were centrifuged, serum recovered, snap frozen and
stored at -70oC.

Total protein content of both samples was determined by Bradford assay,
set to 25, 50, 75, 100, 125, 250, 500 and 1000 (serum only) ug/ml with
bicarboate/carbonate buffer, duplicates of 50 ul were loaded to Nunc
maxisorp plates and incubated at 4°C O/N. We then followed your
‘indirect ELISA protocol’, with both antibodies diluted 1:5000 in 5% non
fat dry milk. For detection, we incubated 30 min in nPP.

As you can clearly see from the attached results, the antibody reacted
well with human serum, but did not show any reaction with the Drosophila
sample.

The AB47455 primary antibody is, as far as we know, the only antibody in
your collection to be tested in ELISA, distinguishing between
constitutive and inducible form of hsp70, and reacting with Drosophila –
with the latter fact not reproducible in our lab and thus of no use for
our work.

Do you have any suggestions for alternative antibodies to detect the
inducible form of Drosophila hsp70 or, maybe, for changes in our
protocol to get the AB47455 working with fruit flies?
Regards,

Read More
Answer

Thank you for taking the time tocontact us and provide this information. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab47455 is tested and covered by our 6 month guarantee for use inELISA and Drosophila samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to help optimize the results.I would also appreciate if you can confirm some further details:

1. Please provide the order numbers and dates of purchase

2. Particularly as the antibody has worked well with human samples, could you confirm if there was any optimization of drosophila sample preparation? Was a lysis buffer used? I can recommend to use a lysis buffer suchas RIPA to provide a suitable protein preparation from the cells in the tissue.

3.I can suggest that perhaps 30 flies may not generate enough protein for detection,although I do not know enough about using Drosophila to make any predictions about this. I can howeversuggest to load 20 - 30 ug of total protein per lane of the gel to ensure thereis enough protein for detection.

This antibody has been tested in ELISA and drosophila separately, but not together. I am sorry we arenot aware of anyone having used this antibody for ELISA with Drosophila before.We know that it works wellin WB for drosophila, andit has been used for a wider range of mammalian ELISAs before. It is possible that the affinity may be less for the fly hsp70, that despite the high degree of homology these are farther away evolutionarily when it comes to insects and that hence there will be a lesser signal.

Finally, there may be factors in the fruit fly that affect the reading, in the same way as serum does in human samples. I am not sure how useful I am being, unfortunately my knowledge about the fruit fly is limited, and the amount of work with this clone in insects over the last 20years is very low, so regrettablythere is little further information to go on.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Read More

Answer

Vielen Dank für Ihre Anfrage und Geduld.

Wie am Telefon besprochen, kann ich bestätigen, dass dieser Antikörper spezifisch für den Hsp70, Hsp72 ist. Falls Sie wieder erwarten ein Problem mit diesem Antikörper hätten, wäre dies auch durch unsere Garantie abgedeckt.

Das Hsp72 ist in der Tat auch in "nicht-gestressten" Zellen vorhanden, welches die Färbung von den HeLa Zellen erklären kann. Diese Expression wurde auch in dem Artikel von Ellis et al beschrieben. http://www.ncbi.nlm.nih.gov/pubmed/10681386

Ich hoffe dies hilft Ihnen weiter. Bitte zögern Sie nicht uns wieder zu kontaktieren, sollten Sie weitere Fragen haben.

Read More

Question
Answer

Thank you for your enquiry. Hsp70 and Hsp72 are the same molecule, this is unfortunately a problem with the hsp nomenclature. Therefore, this product can also detect Hsp72.

Read More
Application
Western blot
Sample
Human Tissue lysate - whole (Peripheral blood mononuclear cells)
Loading amount
20 µg
Specification
Peripheral blood mononuclear cells
Gel Running Conditions
Non-reduced Denaturing (12% Tris-HEPES-SDS)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Mar 09 2009

Application
Western blot
Sample
Mouse Cell lysate - whole cell (NSC34 cell)
Loading amount
10 µg
Specification
NSC34 cell
Gel Running Conditions
Reduced Denaturing (4-12%)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Nov 25 2008

11-17 of 17 Abreviews or Q&A

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