Anti-Hsp70 antibody [2A4]

• 产品名称

  Anti-Hsp70抗体[2A4]
  参阅全部 Hsp70 一抗

• 描述

  小鼠单克隆抗体[2A4] to Hsp70

• 宿主

  Mouse

• 特异性

  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.

• 经测试应用

  适用于: Flow Cyt, WB, IP, ICC/IF, IHC-Pmore details

• 种属反应性

  与反应: Mouse, Human, Saccharomyces cerevisiae
  预测可用于: Cow, Pig

• 免疫原

  Recombinant fragment corresponding to Human Hsp70.
  Database link: P0DMV8

• 表位

  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.

• 阳性对照

• ICC: heat shocked HeLa cells; WB: HeLa, HEK293T, HepG2, A-431, K-562, and MCF7 whole cell lysates.

• 常规说明

  
  

  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

• 形式

  Liquid

• 存放说明

  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.

• 存储溶液

  Preservative: 0.05% Sodium azide
  Constituent: 99% PBS

• 纯度

  Protein A purified

• Primary antibody说明

  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.

• 克隆

  单克隆

• 克隆编号

  2A4

• 同种型

  IgM

The Abpromise guarantee

Abpromise™承诺保证使用ab5442于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。

数据库链接

• 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

• 别名

  hide

• 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

• DnaK type molecular chaperone HSP70 1 antibody

• Epididymis secretory protein Li 103 antibody

• FLJ54303 antibody

• 

  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)

  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 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 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 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 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 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 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 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 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)

  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 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 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 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)

  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.