HSPA8/HSC71/Hsc70 Antibody (13D3)

Western Blot: Hsp70 / Hsc70 Antibody (13D3) [NB120-2788] - Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a HSP70 monoclonal antibody at a dilution of 1:1000 overnight at 4C on a rocking platform, washed in TBS-0.1%Tween 20, and probed with a goat anti-mouse IgM-HRP secondary antibody at a dilution of 1:20,000 for at least 1 hour.

Immunocytochemistry/Immunofluorescence: Hsp70 / Hsc70 Antibody (13D3) [NB120-2788] - Analysis of HSC70 using Monoclonal antibody (13D3) shows staining in NIH-3T3 cells. HSC70 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 HSC70 at a dilution of 1:20-1:200 over night at 4C, washed with PBS and incubated with a DyLight-488 conjugated.

Immunohistochemistry-Paraffin: Hsp70 / Hsc70 Antibody (13D3) [NB120-2788] - Both normal and cancer biopsies of deparaffinized human Prostate carcinoma tissue.

Immunocytochemistry/Immunofluorescence: Hsp70 / Hsc70 Antibody (13D3) [NB120-2788] - 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: Hsp70 / Hsc70 Antibody (13D3) [NB120-2788] - Analysis of HSC70 using HSC70 Monoclonal antibody (13D3) shows staining in HeLa cells. HSC70 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 HSC70 at a dilution of 1:20-1:200 over night at 4C, washed with PBS and incubated with a DyLight-488 conjugated.

Immunocytochemistry/Immunofluorescence: Hsp70 / Hsc70 Antibody (13D3) [NB120-2788] - Analysis of HSC70 using HSC70 Monoclonal antibody (13D3) shows staining in MCF-7 cells. HSC70 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 HSC70 at a dilution of 1:20-1:200 C, washed with PBS and incubated with a DyLight-488 conjugated.

Immunohistochemistry-Paraffin: Hsp70 / Hsc70 Antibody (13D3) [NB120-2788] - Both normal and cancer biopsies of deparaffinized human Tonsil tissue.

Immunohistochemistry-Paraffin: Hsp70 / Hsc70 Antibody (13D3) [NB120-2788] - Both normal and cancer biopsies of deparaffinized human Testis tissue.

Immunoprecipitation: Hsp70 / Hsc70 Antibody (13D3) [NB120-2788] - Analysis of HSC70 was performed on HeLa cells. Antigen-antibody complexes were formed by incubating 500ug whole cell lysate with 2ul of HSC70 monoclonal antibody overnight on a rocking platform at 4C. The immune complexes were captured on 50ul Protein A/G Plus Agarose, washed extensively, and eluted with Lane Marker Reducing Sample Buffer. Samples were 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 probed with a HSC70 monoclonal antibody at a dilution of 1:1000 overnight rotating at 4C, washed in TBST, and probed with goat anti-mouse IgM-HRP secondary antibody for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura.

• Reactivity: Hu, Mu, Rt, Bv, Fe, Fi, Ha, Pm
• Applications: WB, Flow, IB, ICC/IF, IHC, IHC-Fr, IHC-P, IP, B/N
• Clone: 13D3
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated

HSPA8/HSC71/Hsc70 Antibody (13D3) Summary

• Immunogen: Mouse spermatogenic cell protein.
• Specificity: This displays slight cross-reactivity to HSP 70.
• Isotype: IgM
• Clonality: Monoclonal
• Host: Mouse
• Gene: HSPA8
• Purity: Unpurified

Applications/Dilutions

• Dilutions:
  • Western Blot 1:1000 - 1:5000
  • Flow Cytometry 1:10 - 1:1000
  • Immunoblotting
  • Immunocytochemistry/Immunofluorescence 1:20 - 1:200
  • Immunohistochemistry 1:20 - 1:200
  • Immunohistochemistry-Frozen 1:20 - 1:200
  • Immunohistochemistry-Paraffin 1:20 - 1:200
  • Immunoprecipitation 2 ul
  • Block/Neutralize
• Application Notes: WB: Detects an approx. 70 kDa protein representing Hsc 70. By 2D gel electrophoresis, this antibody binds strongly to Hsc 70 and faintly to HSP 70 both before and after heat shock. IHC: Staining of Hsc 70 in mouse spermatids/spermatozoa results in staining restricted to the post acrosomal region in condensing spermatids and to the midpiece in spermatozoa. May be useful in IHC-Fr. Use in Immunoblotting reported in scientific literature (PMID 28095639).
• Theoretical MW: 70 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Reactivity Notes

Reported cross-reactivity with Bovine in literature (Terlecky et al.). Hamster reactivity reported in scientific literature (PMID: 16737757). Fish reactivity reported in scientific literature (PMID: 9006893). Rat reactivity reported by customer review. Please note that this antibody is reactive to Mouse and derived from the same host, Mouse. Additional Mouse on Mouse blocking steps may be required for IHC and ICC experiments. Please contact Technical Support for more information.

Packaging, Storage & Formulations

• Storage: Store at -20C. Avoid freeze-thaw cycles.
• Buffer: Ascites diluted with PBS
• Preservative: 0.05% Sodium Azide
• Purity: Unpurified

Alternate Names for HSPA8/HSC71/Hsc70 Antibody (13D3)

• Heat shock 70 kDa protein 8
• heat shock 70kD protein 8
• heat shock 70kDa protein 8
• heat shock cognate 71 kDa protein
• heat shock cognate protein 54
• heat shock cognate protein, 71-kDa
• HSC54
• HSC70
• HSC70heat shock 70kd protein 10
• HSC71
• HSP71
• HSP73
• HSP73constitutive heat shock protein 70
• HSPA10
• HSPA8
• LAP1
• lipopolysaccharide-associated protein 1
• LPS-associated protein 1
• MGC131511
• MGC29929
• NIP71
• N-myristoyltransferase inhibitor protein 71

Background

Heat shock proteins (HSP's) or stress response proteins (SRP's) are synthesized in variety of environmental and pathophysiological stressful conditions. Many HSP's are involved in processes such as protein denaturation-renaturation, folding-unfolding, transport-translocation, activation-inactivation, and secretion. HSP70 is found to be associated with steroid receptors,actin, p53, polyoma T antigen, nucleotides, and other unknown proteins. Also, HSP70 has been shown to be involved in protective roles against thermal stress, cytotoxic drugs, and other damaging conditions.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for HSPA8/HSC71/Hsc70 Antibody (NB120-2788)(16)

Publications using NB120-2788

• Juste YR, Cuervo AM. Analysis of Chaperone-Mediated Autophagy. Methods Mol. Biol. Jan 6 2019 [PMID: 30610733] (ICC/IF, Rat)
• Seki T, Sato M, Kibe Y et al. Lysosomal dysfunction and early glial activation are involved in the pathogenesis of spinocerebellar ataxia type 21 caused by mutant transmembrane protein 240. Neurobiol. Dis. Sep 2 2018 [PMID: 30184469] (WB, Human, Rat, Mouse)
• Pajares M, Rojo AI, Arias E et al. Transcription factor NFE2L2/NRF2 modulates chaperone-mediated autophagy through the regulation of LAMP2A Autophagy Jul 26 2018 [PMID: 29950142] (WB, Human)
• Gong Z, Tasset I, Diaz A et al. Humanin is an endogenous activator of chaperone-mediated autophagy J. Cell Biol. Feb 5 2018 [PMID: 29187525] (WB, Rat)
• Zhang S, Eitan E, Mattson MP. Early Involvement of Lysosome Dysfunction in the Degeneration of Cerebral Cortical Neurons Caused by the Lipid Peroxidation Product 4-Hydroxynonenal J. Neurochem Jan 17 2017 [PMID: 28095639] (IB, Rat)
• Ganesan S, Reynolds C, Hollinger K et al. Twelve hours of heat stress induces inflammatory signaling in porcine skeletal muscle. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2016 Mar 23 [PMID: 27009052]
• Park C, Suh Y, Cuervo AM. Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage Nat Commun 2015 Apr 17 [PMID: 25880015] (WB, ICC/IF, IP, Mouse)
• Kett LR, Stiller B, Bernath MM et al. alpha-Synuclein-Independent Histopathological and Motor Deficits in Mice Lacking the Endolysosomal Parkinsonism Protein Atp13a2 J. Neurosci. 2015 Apr 08 [PMID: 25855184]
• Ferreira JV, Fofo H, Bejarano E et al. STUB1/CHIP is required for HIF1A degradation by chaperone-mediated autophagy. Autophagy 2013 Jun 7 [PMID: 23880665]
• Hubbi ME, Hu H, Kshitiz F et al. Chaperone-mediated autophagy targets HIF-1alpha for lysosomal degradation. J Biol Chem 2013 Mar 1 [PMID: 23457305]
• Orenstein SJ, Kuo SH, Tasset I et al. Interplay of LRRK2 with chaperone-mediated autophagy. Nat Neurosci 2013 Apr [PMID: 23455607] (WB, Human)
• H Koga, S KaushikA M Cuervo. Altered lipid content inhibits autophagic vesicular fusion. FASEB J; fj09-144519. 2010 [PMID: 20375270]
• Guerrero CA, Bouyssounade D, Zarate S et al. Heat shock cognate protein 70 is involved in rotavirus cell entry. J Virol;76(8):4096-102. 2002 Apr. [PMID: 11907249] (FLOW)
• Polanowska-Grabowska R, Simon CG Jr, Falchetto R, Shabanowitz J, Hunt DF, Gear AR. Platelet adhesion to collagen under flow causes dissociation of a phosphoprotein complex of heat-shock proteins and protein phosphatase 1. Blood;90(4):1516-26. 1997 Aug 15. [PMID: 9269769] (IP)
• Maekawa M, O'Brien DA, Allen RL, Eddy EM. Heat-shock cognate protein (hsc71) and related proteins in mouse spermatogenic cells. Biol Reprod;40(4):843-52. 1989 Apr. [PMID: 2665832] (ICC/IF)
• Terlecky SR, Chiang HL, Olson TS, Dice JF. Protein and peptide binding and stimulation of in vitro lysosomal proteolysis by the 73-kDa heat shock cognate protein. J Biol Chem;267(13):9202-9. 1992 May 5. [PMID: 1577755] (WB)

Reviews for HSPA8/HSC71/Hsc70 Antibody (NB120-2788) (2)

Average Rating: 3.5

(Based on 2 reviews)

We have 2 reviews tested in 2 species: Human, Mouse.

reviewed by: Anonymous Simple Western Mouse 01/21/2016

Summary

• Application: Simple Western
• Sample Tested: whole brain homogenates from mice
• Species: Mouse

Blocking

• Blocking Details: n/a

Primary Anitbody

• Dilution Ratio: 1:2000, 1:500, 1:100

Details

• Detection Notes: WES Simple Western platform

Comments

• Comments: It works better at low concentrations.

reviewed by: Anonymous WB Human 02/04/2009

Summary

• Application: Western Blot
• Sample Tested: HEK293 cell lysate, Sample Amount: 20 ug
• Species: Human
• Lot: 056-114

Blocking

• Blocking Details: Blocking Buffer: Li-Cor blocking buffer, Blocking Time: 30 minutes, Blocking Temp: Room temperature

Primary Anitbody

• Dilution Ratio: Dilution Ratio: 1/10000, Incubation Dilution Buffer: Li-Cor blocking buffer, Incubation Time: overnight, Incubation Temp: 4C

Secondary Antibody

• Secondary Description: Secondary Ab: Alexa 680 goat anti-mouse pAb, Secondary Ab Dilution Ratio: 1/25000

Details

• Detection Notes: Detection Method: Infra-red imaging, Specific Bands: 70 kDa, Non-Specific Bands: 40 kDa

FAQs for HSPA8/HSC71/Hsc70 Antibody (NB120-2788). (Showing 1 - 2 of 2 FAQs).

1. What is the concentration of this product?
   • As this product is sold as unpurified ascites, the concentration of the antibody has not been determined.
2. Would any endotoxin and azide exist in buffer?
   • The antibody solution contains 0.05% sodium azide. We have not tested this product for endotoxin levels.

Bioinformatics

• Gene Symbol: HSPA8
• Uniprot:
  • P08107()