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    hsp70-antibody-2a4-ab5442.pdf

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Signal Transduction Protein Trafficking Chaperones Heat Shock Proteins
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Anti-Hsp70 antibody [2A4] (ab5442)

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Reviews (1)Q&A (3)References (15)

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Western blot - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunoprecipitation - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
  • Flow Cytometry - Anti-Hsp70 antibody [2A4] (ab5442)

Key features and details

  • Mouse monoclonal [2A4] to Hsp70
  • Suitable for: Flow Cyt, WB, IP, ICC/IF, IHC-P
  • Reacts with: Mouse, Human, Saccharomyces cerevisiae
  • Isotype: IgM

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Overview

  • Product name

    Anti-Hsp70 antibody [2A4]
    See all Hsp70 primary antibodies
  • Description

    Mouse monoclonal [2A4] to Hsp70
  • Host species

    Mouse
  • Specificity

    ab5442 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 applications

    Suitable for: Flow Cyt, WB, IP, ICC/IF, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Human, Saccharomyces cerevisiae
    Predicted to work with: Cow, Pig
  • Immunogen

    Recombinant fragment corresponding to Human Hsp70.
    Database link: P0DMV8

  • Epitope

    Epitope 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; WB: HeLa, HEK293T, HepG2, A-431, K-562, and MCF7 whole cell lysates.
  • General notes

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

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 or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer

    Preservative: 0.05% Sodium azide
    Constituent: 99% PBS
  • Concentration information loading...
  • Purity

    Protein 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 number

    2A4
  • Isotype

    IgM
  • Research areas

    • Signal Transduction
    • Protein Trafficking
    • Chaperones
    • Heat Shock Proteins
    • Cancer
    • Tumor biomarkers
    • Other
    • Neuroscience
    • Processes

Associated products

  • Compatible Secondaries

    • Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
    • Goat Anti-Mouse IgM mu chain (DyLight® 488) (ab97007)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Mouse IgM [B11/7] - Isotype control (ab91545)
  • Recombinant Protein

    • Recombinant human Hsp70 protein (Active) (ab78434)

Applications

The Abpromise guarantee

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 Abreviews Notes
Flow Cyt
Use 1µg for 106 cells.

ab91545 - Mouse monoclonal IgM, is suitable for use as an isotype control with this antibody.

 

WB (1)
Use a concentration of 1 µg/ml. Detects a band of approximately 70 kDa (predicted molecular weight: 70 kDa).
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).

Notes
Flow Cyt
Use 1µg for 106 cells.

ab91545 - Mouse monoclonal IgM, is suitable for use as an isotype control with this antibody.

 

WB
Use a concentration of 1 µg/ml. Detects a band of approximately 70 kDa (predicted molecular weight: 70 kDa).
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).

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

    • Entrez Gene: 281825 Cow
    • Entrez Gene: 15511 Human
    • Entrez Gene: 3303 Human
    • Entrez Gene: 193740 Mouse
    • Entrez Gene: 3304 Mouse
    • Entrez Gene: 396906 Pig
    • Omim: 140550 Human
    • Omim: 603012 Human
    • SwissProt: Q27975 Cow
    • SwissProt: P0DMV8 Human
    • SwissProt: P0DMV9 Human
    • SwissProt: P17879 Mouse
    • SwissProt: Q61696 Mouse
    • SwissProt: P34930 Pig
    see all
  • 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
    • HSP70-2 antibody
    • HSP70.1 antibody
    • HSP70.1/HSP70.2 antibody
    • HSP71_HUMAN antibody
    • HSP72 antibody
    • HSPA1 antibody
    • HSPA1A antibody
    • HSPA1B antibody
    see all

Images

  • Western blot - Anti-Hsp70 antibody [2A4] (ab5442)
    Western blot - Anti-Hsp70 antibody [2A4] (ab5442)
    All lanes : Anti-Hsp70 antibody [2A4] (ab5442) at 1 µg/ml

    Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
    Lane 2 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
    Lane 3 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
    Lane 4 : A431 (human epidermoid carcinoma cell line) whole cell lysate
    Lane 5 : K562 (human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
    Lane 6 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate

    Lysates/proteins at 30 µg per lane.

    Secondary
    All lanes : Goat anti-Mouse IgG H+L (HRP) at 1/4000 dilution

    Predicted band size: 70 kDa
    Observed band size: 70 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)

    IHC image of Hsp70 staining in human lung formalin fixed paraffin embedded tissue section*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab5442, 1/2000 dilution overnight at +4°C. An HRP-conjugated secondary (ab97230, 1/2000 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

    The inset negative control image is secondary-only at 1/500 dilution.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)

    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 - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)

    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 - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)

    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 - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)

    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 - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)

    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 - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)

    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 - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunoprecipitation - Anti-Hsp70 antibody [2A4] (ab5442)

    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 - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [2A4] (ab5442)

    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
    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 (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
    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 (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
    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 (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [2A4] (ab5442)
    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.

  • Flow Cytometry - Anti-Hsp70 antibody [2A4] (ab5442)
    Flow Cytometry - Anti-Hsp70 antibody [2A4] (ab5442)
    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.

Protocols

  • Flow cytometry protocols
  • Immunoprecipitation protocols
  • Immunohistochemistry protocols
  • Immunocytochemistry & immunofluorescence protocols

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (15)

Publishing research using ab5442? Please let us know so that we can cite the reference in this datasheet.

ab5442 has been referenced in 15 publications.

  • Szekely L  et al. Pulmonary stromal expansion and intra-alveolar coagulation are primary causes of COVID-19 death. Heliyon 7:e07134 (2021). PubMed: 34056141
  • Abd El-Baset SA  et al. Mesna ameliorates acute lung injury induced by intestinal ischemia-reperfusion in rats. Sci Rep 11:13356 (2021). PubMed: 34172794
  • Wang Q  et al. Systematic Transcriptome Analysis of Noise-Induced Hearing Loss Pathogenesis Suggests Inflammatory Activities and Multiple Susceptible Molecules and Pathways. Front Genet 11:968 (2020). PubMed: 33005175
  • Son YO  et al. RNA-binding protein ZFP36L1 regulates osteoarthritis by modulating members of the heat shock protein 70 family. Nat Commun 10:77 (2019). PubMed: 30622281
  • Khafaga AF  et al. The adaptogenic anti-ageing potential of resveratrol against heat stress-mediated liver injury in aged rats: Role of HSP70 and NF-kB signalling. J Therm Biol 83:8-21 (2019). PubMed: 31331528
View all Publications for this product

Customer reviews and Q&As

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1-4 of 4 Abreviews or Q&A

Western blot abreview for Anti-Hsp70 antibody [2A4]

Good
Abreviews
Abreviews
abreview image
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%
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Jan 31 2007

Question

High background in IHC-P on human liver sections

Read More

Abcam community

Verified customer

Asked on Nov 14 2012

Answer

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 I can help you with, or if you have any issues with these antibodies, please let me know.

Read More

Abcam Scientific Support

Answered on Nov 14 2012

Question

Recently i bought AB 5442 antibodies, I need help to know molecular weight and PI of this product. I am planning to bind color microsphers, if you can help me to suggest buffer for binding sphers will be great, i am also looking for storage buffer after binding. PI of protein is important for selection of buffers if you have not tested this batch then how about your earlier batch, what will be PI of this product. I understand this is not new product on your list. There must be some QC data

Read More

Abcam community

Verified customer

Asked on Oct 14 2011

Answer

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. As the on-line datasheet demonstrates, ab5442 has not been tested in kinetic studies (only in immunotechniques such as ICC/IF, IHC-P, WB ) neither in binding spheres with our antibodies in the lab. Apologies!

Read More

Abcam Scientific Support

Answered on Oct 14 2011

Question

I am the technical support specialist of Jingmei Biotech Co. Ltd. , China. One of our customers met with some difficulties in his IHC-P experiments. He ordered the product Cat#ab5442, Hsp70 antibody,but the repeated results showed that all tissues (human retinoblastoma and rat eyeball slides)were stained in the IHC-P inculding connective tissue and collagen tissue (while the negative control had no brown staining). In brief , his protocol is: (1) Deparaffinization and rehydration routinely. (2) Wash sections three time in PBS for 5 minutes each. (3) Antigen unmasking and retrieval: Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0 then maintain at a sub-boiling temperature for 5-8 minutes, Cool slides on bench top for 30 minutes. (4) Wash sections three time in PBS for 5 minutes each. (5) Staining: 1) Incubate sections in 0.5-3% hydrogen peroxide for 10 minutes, then wash sections three time in PBS for 5 minutes each. 2) Block each section with normal goat serum (according to the second antibody) for 20 minutes at room temperature . 3) Without washing, remove normal goat serum and add diluted primary antibody (Hsp70 antibody, Cat#ab5442)to each section (dilute antibody in blocking solution.) , incubate overnight at 4°C or 1-2 hour at 37°C . 4) Wash sections three time in PBS for 5 minutes each. 5) Add diluted HRP-conjugated goat anti-mouse second antibody to each section , incubate 30 minutes at 37°C . 6) Wash sections three time in PBS for 5 minutes each. 7) Add 0.04%DAB + 0.03% hydrogen peroxide for 3-10 minutes and monitor staining closely. 8) Wash sections three time in PBS for 5 minutes each. 9) Counterstain sections in hematoxylin. 10) Mount coverslips. He had repeated the IHC experiments for about 10 times with various conditions,but the results were similar. He made efforts as follows: (1) Reduced the concentration of primary antibody and second antibody. (2) Decreased the incubation time of antibody. (3) Reduced the concentration of DAB. (4) Decreased the coloration time of DAB. (5) Increased the concentration of hydrogen peroxide and replace the hydrogen peroxide as new one. (6) prolonged the block time with serum. (7) prolonged the washing time. (8) Added 0.1% Tween 20 in washing Buffer. [ The attachments showed the results of IHC-P: PIC1-PIC2 showed the staining results using the nomal procedure (see above),PIC3 showed the negative control staining result in which the primary antibody was replaced with PBS. ] I and he are puzzled with it . Could you please have some effective suggestion for him? Or could you please give him a replacement?

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Abcam community

Verified customer

Asked on Sep 02 2006

Answer

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 laboratory that a heat mediated antigen retrieval method with citrate buffer was adequate and that a dilution of 1:100 should work. Is this the sort of range your customer has tried? Can you please also provide your order details? Thank you for confirming if the problem could be solved by using a more concentrated form of the antibody or if this option has been tried,

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Abcam Scientific Support

Answered on Sep 05 2006

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