Anti-Hsp70 antibody [5A5]

• 产品名称

  Anti-Hsp70抗体[5A5]
  参阅全部 Hsp70 一抗

• 描述

  小鼠单克隆抗体[5A5] to Hsp70

• 宿主

  Mouse

• 经测试应用

  适用于: IHC-P, WB, ICC/IF, IP, Flow Cytmore details

• 种属反应性

  与反应: Mouse, Rat, Dog, Human, African green monkey
  

• 免疫原

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

• 表位

  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 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.

• 阳性对照

• WB: HEK-293, A549, IMR-32, Jurkat, Jurkat treated with heat shock , MDCK, NIH/3T3, PC-12, COS-7, HeLa, K562, A431, U-2 OS whole cell and mouse ovary tissue lysate. IHC-P: Human prostate, tonsil and testis tissue. ICC: HeLa, NIH- 3T3, U251 MG, C6 cells. IP: HeLa 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. Avoid freeze / thaw cycle.

• 存储溶液

  Preservative: 0.05% Sodium azide
  Constituents: PBS, BSA

• 纯度

  Protein A purified

• 克隆

  单克隆

• 克隆编号

  5A5

• 同种型

  IgG1

The Abpromise guarantee

Abpromise™承诺保证使用ab2787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: 403612 Dog

• Entrez Gene: 15511 Human

• Entrez Gene: 3303 Human

• Entrez Gene: 193740 Mouse

• Entrez Gene: 3304 Mouse

• Entrez Gene: 24472 Rat

• Entrez Gene: 294254 Rat

• Omim: 140550 Human

• Omim: 603012 Human

• SwissProt: Q7YQC6 Dog

• SwissProt: P0DMV8 Human

• SwissProt: P0DMV9 Human

• SwissProt: P17879 Mouse

• SwissProt: Q61696 Mouse

• SwissProt: P0DMW0 Rat

• SwissProt: P0DMW1 Rat

别名

• 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

• 

  Western blot - Anti-Hsp70 antibody [5A5] (ab2787)

  All lanes : Anti-Hsp70 antibody [5A5] (ab2787) at 1/500 dilution
  
  Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
  Lane 2 : A549 (Human lung carcinoma cell line) whole cell lysate
  Lane 3 : IMR32 (Human brain neuroblast cell line) whole cell lysate
  Lanes 4-5 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
  Lane 6 : MDCK (Canine kidney cell line) whole cell lysate
  Lane 7 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate
  Lane 8 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
  Lane 9 : COS-7 (African green monkey kidney fibroblast-like cell line) whole cell lysate
  Lane 10 : Mouse ovary tissue lysate
  
  Lysates/proteins at 30 µg per lane.
  
  Secondary
  All lanes : Goat anti-mouse IgG (H+L) HRP at 1/4000 dilution
  
  Developed using the ECL technique.

Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [5A5] (ab2787)

Immunofluorescent analysis of U-251 MG (Human brain glioma cell line) cells labeling Hsp70 (green) with ab2787. 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [5A5] (ab2787)

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.

• 

  Flow Cytometry - Anti-Hsp70 antibody [5A5] (ab2787)

  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.

• 

  Immunoprecipitation - Anti-Hsp70 antibody [5A5] (ab2787)

  Immunoprecipitation of Hsp70 was performed on HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate. 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.

• 

  Western blot - Anti-Hsp70 antibody [5A5] (ab2787)

  All lanes : Anti-Hsp70 antibody [5A5] (ab2787) at 1/1000 dilution
  
  Lane 1 : HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
  Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
  Lane 3 : K562 (Human chronic myelogenous leukemia cell line from bone marrow ) whole cell lysate
  Lane 4 : A431 (Human epidermoid carcinoma cell line) whole cell lysate
  Lane 5 : U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate
  
  Lysates/proteins at 50 µg per lane.
  
  Secondary
  All lanes : Goat anti-mouse IgG HRP secondary antibody at 1/20000 dilution
  
  
  

  Western blot analysis of Hsp70 was performed by 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 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween 20, and incubated with secondary antibody for at least 1 hour. Chemiluminescent detection was performed.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [5A5] (ab2787)

  Immunofluorescent analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Hsp70 (green) with ab2787. 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.
  
  

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [5A5] (ab2787)

  Immunofluorescent analysis of C6 (Rat glial tumor cell line) cells labeling Hsp70 (green) with ab2787. 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.
  
  

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [5A5] (ab2787)

  Immunofluorescent analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) and NIH/3T3 (Mouse embryo fibroblast cell line) cells labeling Hsp70 (green) with ab2787. 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 a HSP70 Monoclonal Antibody, at a dilution of 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat-anti-mouse IgG secondary antibody at a dilution of 1/400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [5A5] (ab2787)Image from Park R et al, J Biol Chem. 2011 Mar 18;286(11):9748-62. Epub 2011 Jan 13, Fig 4. DOI 10.1074/jbc.M110.198325

  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.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [5A5] (ab2787)

  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 (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [5A5] (ab2787)

  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.
  
  

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [5A5] (ab2787)

  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.

• 

  Western blot - Anti-Hsp70 antibody [5A5] (ab2787)This image is courtesy of an anonymous Abreview

  Western blot analysis of U2OS cell lysate (30μg/lane) labelling Hsp70 with ab2787 at 1/1000 in 5% milk in TBST for 13 hours at 4°C. A IRDye® 800-conjugated Goat anti-Mouse polyclonal (1/10000) was used as the secondary antibody.

  See Abreview