HSP70-17 Antibody

What is HSP70 and why are antibodies against it important in research?

HSP70 (Heat Shock Protein 70) represents one of the most ubiquitous classes of molecular chaperones and is highly conserved across organisms. The 70-kDa heat shock protein family controls all aspects of cellular proteostasis, including nascent protein chain folding, protein import into organelles, recovery of proteins from aggregation, and assembly of multi-protein complexes . These chaperones enhance organismal survival and longevity under proteotoxic stress by facilitating cell viability and protein damage repair .

Antibodies against HSP70 are essential research tools that enable:

• Detection and quantification of HSP70 expression across tissues and cell types

• Investigation of HSP70's roles in normal cellular function and disease states

• Examination of protein-protein interactions involving HSP70

• Study of subcellular localization in both normal and pathological conditions

The HSP70 family consists of multiple members with diverse functions. Current literature has identified members up to HSP70-14, with HSP70-14 (HspA14, Hsp70L1) being derived from human dendritic cells and having potent adjuvant effects that polarize responses towards Th1 .

What applications are HSP70 antibodies commonly used for in laboratory research?

HSP70 antibodies serve as versatile tools in laboratory research with multiple validated applications:

Methodological considerations for optimal results:

• Positive control: 1 μg/ml of anti-HSP70 antibody has been validated for detection in 20 μg of heat-shocked HeLa cell lysate using colorimetric immunoblot analysis with Goat anti-mouse IgG:HRP as the secondary antibody

• Cross-reactivity: Some antibodies like clone C92F3A-5 have been specifically validated not to cross-react with the constitutive HSC70 (HSP73)

• Species reactivity: Many HSP70 antibodies demonstrate broad cross-reactivity across species including human, mouse, rat, and even non-mammalian species like C. elegans and Drosophila melanogaster

How do I validate the specificity of an HSP70 antibody for my experimental applications?

Rigorous validation of antibody specificity is critical for reliable research results. For HSP70 antibodies, implement these methodological approaches:

• Western blot analysis:

  • Confirm detection of the expected ~70 kDa band in positive control samples (e.g., heat-shocked HeLa cells)

  • Compare with negative controls (untreated samples or knockdown cells)

  • Assess potential cross-reactivity with other HSP family members

• Genetic validation:

  • Perform siRNA knockdown experiments targeting HSP70

  • Western blot analysis following knockdown should show decreased HSP70 signal

  • Example: HSP70 siRNA interference on HEL cell line resulted in decreased expression levels of HSP70, JAK2, and p-STAT5 as confirmed by western blot

• Pharmacological validation:

  • Treat samples with HSP70 inhibitors like KNK437 at appropriate concentrations (10-100 μM)

  • Confirm decreased HSP70 expression by western blot

  • Molecular effects of KNK437 in HEL cell line show decreased HSP70 expression along with p-JAK2

• Functional assays:

  • Perform immunoprecipitation followed by mass spectrometry to confirm pull-down of known HSP70 interacting partners

  • Compare results with published interaction data

• Cross-reactivity testing:

  • Test against recombinant HSP70 family members

  • Utilize antibodies like clone C92F3A-5 that have been verified not to cross-react with HSC70 (HSP73)

A comprehensive validation protocol increases confidence in experimental results and helps troubleshoot unexpected findings.

What is the significance of HSP70 autoantibodies in autoimmune disease research?

HSP70 autoantibodies have emerged as important factors in autoimmune disease pathogenesis and potential therapeutic targets:

• Disease association:

  • Circulating anti-HSP70 autoantibodies have been detected in patients with Epidermolysis Bullosa Acquisita (EBA) compared to healthy controls

  • These autoantibodies may serve as biomarkers of disease activity or progression

• Pathological mechanisms:

  • In experimental EBA, anti-HSP70 IgG administration aggravated disease severity, with higher clinical scores and more extensive lesions compared to isotype control-treated mice

  • Dermal neutrophil infiltration was significantly higher in anti-HSP70 IgG-treated mice

  • Mechanistically, increased NF-κB activation was observed in the skin of anti-HSP70 IgG-treated mice with EBA

• Research methodologies:

  • ELISA assays can effectively measure levels of circulating anti-HSP70 autoantibodies in patient sera

  • Antibody transfer models enable study of the pathogenic effects of anti-HSP70 antibodies in vivo

  • Histological analysis can quantify neutrophil infiltration and tissue damage

• Potential therapeutic relevance:

  • The NF-κB signaling pathway activated by anti-HSP70 autoantibodies may represent a possible therapeutic target for autoimmune diseases

  • Understanding the dual role of HSP70 in autoimmunity could lead to novel therapeutic approaches

These findings highlight the complex interplay between HSP70, autoantibodies, and immune pathways in autoimmune disease pathogenesis, suggesting several avenues for further research.

How do anti-HSP70 antibodies affect T-cell differentiation and function in experimental models?

Anti-HSP70 antibody treatment has demonstrated significant effects on T-cell populations with implications for immunomodulation:

• Impact on T-cell subsets:

  • Anti-HSP70 antibody treatment significantly decreases the percentage of pro-inflammatory Th17 cells (CD4+IL-17+) in splenic tissue

  • Treatment is associated with a significant increase in the CD4+FoxP3+:Th17 ratio, indicating a shift from inflammatory to regulatory balance

  • Interestingly, the treatment shows no significant effect on splenic CD4+FoxP3+ regulatory T-cell frequencies or blood CD4+CD25+ cell populations

• Methodological approach:

  • Flow cytometry analysis using specific markers:

    • CD4+FoxP3+ cells for regulatory T cells

    • CD4+CD25+ cells for activated/regulatory T cells

    • CD4+IL-17+ cells for Th17 cells

  • Calculation of CD4+FoxP3+:Th17 ratio as a key immunoregulatory metric

  • Comparison between anti-HSP70 antibody treatment and isotype controls

• Experimental data:

  T-cell Population	Effect of Anti-HSP70 Treatment	Statistical Significance
  Splenic CD4+FoxP3+	No significant change	p > 0.05
  Blood CD4+CD25+	No significant change	p > 0.05
  Splenic CD4+IL-17+ (Th17)	Significant decrease	p < 0.05
  CD4+FoxP3+:Th17 ratio	Significant increase	p < 0.05

• Implications for immunotherapy:

  • The ability to selectively reduce pro-inflammatory Th17 cells without affecting regulatory T cells suggests potential for targeted immunomodulation

  • This effect could be particularly relevant for autoimmune diseases with Th17-driven pathology

  • The mechanisms underlying this selective modulation warrant further investigation

These findings provide important insights into how targeting HSP70 might be leveraged for therapeutic immunomodulation in conditions with excessive inflammatory T-cell responses .

What experimental models are optimal for studying HSP70 antibody effects in immune-mediated diseases?

Several experimental models have been validated for investigating HSP70 antibody effects in immune-mediated diseases:

• Antibody transfer-induced Epidermolysis Bullosa Acquisita (EBA) model:

  • Methodology: Anti-COL7 antibodies are administered to induce neutrophil infiltration and blister formation

  • Assessment parameters:

    • Clinical scores (percentage of body surface area with lesions)

    • Histological analysis of neutrophil infiltration

    • Immunofluorescence for immune complex deposition

    • Measurement of circulating anti-HSP70 IgG and IFN-γ levels by ELISA

  • Key findings: EBA induction was paralleled by generation of circulating anti-HSP70 IgG and elevated blood IFN-γ levels. Anti-HSP70 IgG administration exacerbated disease severity compared to isotype controls

• Imiquimod (IMQ)-induced skin inflammation model:

  • Methodology: Topical application of IMQ induces psoriasis-like inflammation

  • Assessment parameters:

    • Flow cytometric analysis of T-cell populations

    • Measurement of inflammatory cytokines

    • Histological evaluation of skin inflammation

  • Key findings: Anti-HSP70 antibody treatment decreased pro-inflammatory Th17 cells and increased the CD4+FoxP3+:Th17 ratio

• Cell culture models:

  • HEL and Ba/F3 JAK2 V617F cell lines:

    • Used to study effects of HSP70 inhibition via KNK437 and siRNA

    • Viability assays assess functional outcomes

    • Western blotting measures effects on signaling pathways

  • Key findings: HSP70 inhibition decreased cell viability and reduced signaling through JAK2, ERK, and STAT5 pathways

• Design considerations for HSP70 antibody experiments:

  • Include appropriate controls (isotype antibodies)

  • Determine optimal antibody dosing and timing

  • Consider potential differences between prophylactic (pre-disease) versus therapeutic (established disease) administration

  • Account for species differences in HSP70 response

These models provide complementary approaches for understanding HSP70's role in immune regulation and validating therapeutic strategies targeting this pathway.

How can researchers distinguish between different members of the HSP70 family in experimental settings?

Distinguishing between different HSP70 family members is crucial for understanding their specific functions. Here are methodological approaches:

• Antibody-based discrimination:

  • Select antibodies with validated specificity for particular HSP70 family members

  • Example: Clone C92F3A-5 has been confirmed not to cross-react with HSC70 (HSP73)

  • Western blot analysis can confirm detection of the expected molecular weight band

• Expression pattern analysis:

  • Inducible HSP70 (HSPA1A/HSPA1B) versus constitutive HSC70 (HSPA8):

    • Compare unstressed cells (primarily expressing constitutive forms) with stressed cells (upregulating inducible forms)

    • Heat shock treatment (42-43°C for 30-60 minutes) induces HSPA1A/B but not HSPA8

    • KNK437 (HSP inhibitor) treatment selectively inhibits inducible forms

• Genetic manipulation approaches:

  • siRNA targeting specific HSP70 family members

  • CRISPR/Cas9 gene editing to tag or knockout specific variants

  • Overexpression of individual family members with epitope tags

• Tissue and subcellular localization:

  • Different HSP70 family members have distinct subcellular distributions:

    • HSPA5 (BiP/GRP78): Endoplasmic reticulum

    • HSPA9 (mortalin/GRP75): Primarily mitochondrial

    • HSPA13 (Stch): Microsome-associated

  • Immunofluorescence with specific antibodies can reveal these patterns

• Functional assays:

  • ATP binding and hydrolysis activities may differ

  • Co-chaperone interactions can be specific to certain family members

  • Client protein binding affinities vary between family members

Understanding these distinctions is essential for accurately interpreting experimental results involving HSP70 family members.

What are the current methodologies for studying HSP70's role in cancer using antibody-based approaches?

HSP70 has been linked to several carcinomas, with its expression associated with therapeutic resistance, metastasis, and poor clinical outcomes . Here are current methodologies for investigating this relationship:

• Expression profiling in tumor tissues:

  • Immunohistochemistry (IHC): Quantify HSP70 expression patterns in tumor versus normal tissues

    • Recommended dilution: 1:10000 for IHC applications

    • Compare expression with clinical outcomes and tumor characteristics

  • Tissue microarray analysis: High-throughput screening across multiple tumor samples

  • Multiplex immunofluorescence: Co-localization with other cancer markers

• Functional studies in cancer cell lines:

  • siRNA knockdown with antibody validation:

    • Transfect cancer cells with HSP70-specific siRNA

    • Confirm knockdown using Western blot with anti-HSP70 antibodies

    • Assess effects on proliferation, apoptosis, and drug resistance

  • HSP70 inhibition studies:

    • Treat cancer cells with inhibitors like KNK437

    • Monitor effects on HSP70 expression and cancer cell viability

    • Example: KNK437 treatment (50 μM) reduced viability in HEL and Ba/F3 JAK2 V617F cell lines

• Mechanistic investigation:

  • Co-immunoprecipitation with HSP70 antibodies:

    • Identify cancer-specific HSP70 client proteins and complexes

    • Elucidate pathways dependent on HSP70 in malignant cells

  • ChIP assays: Study HSP70's role in transcriptional regulation

  • Proximity ligation assays: Detect HSP70 interactions in situ

• HSP70 in therapy resistance:

  • Pre/post-treatment expression analysis:

    • Compare HSP70 levels before and after chemotherapy or radiation

    • Correlate expression changes with treatment response

  • Combination treatment strategies:

    • Test HSP70 inhibitors in combination with standard cancer therapies

    • Use antibodies to monitor HSP70 expression/activity

• HSP70 as a potential therapeutic target:

  • Antibody-drug conjugates: Target HSP70-overexpressing cancer cells

  • Aptamer-based approaches: Develop HSP70-targeting therapeutic molecules

  • Small molecule inhibitor development: Use antibodies to validate target engagement

These methodologies help elucidate how HSP70 protects cancer cells from proteotoxic stress, suppresses cellular senescence, and confers resistance to stress-induced apoptosis including protection against cytostatic drugs and radiation therapy .

What are the optimal experimental conditions for detecting extracellular HSP70 using antibody-based methods?

Extracellular HSP70 has important immunomodulatory functions, either facilitating cross-presentation of immunogenic peptides via MHC or acting as "chaperokines" to stimulate innate immune responses . Detecting extracellular HSP70 requires specific methodological considerations:

• Sample collection and processing:

  • Biological fluids:

    • Collect blood/serum samples using standardized protocols

    • Process promptly (within 1-2 hours) to prevent artifactual HSP70 release

    • Centrifuge at 2000-3000g for 10-15 minutes to remove cells

    • Consider additional high-speed centrifugation (10,000g) to remove microvesicles

  • Cell culture supernatants:

    • Culture cells in serum-free media to avoid bovine HSP70 contamination

    • Include both stressed and unstressed conditions

    • Filter supernatants (0.22μm) to remove cellular debris

• ELISA-based detection:

  • Commercial kits versus customized assays:

    • Use validated antibody pairs with one capture and one detection antibody

    • Typical sensitivity range: 0.2-10 ng/ml

  • Protocol optimization:

    • Sample dilution series to ensure measurements within linear range

    • Include recombinant HSP70 standards (0.1-100 ng/ml)

    • Control for potential interfering factors in biological samples

• Flow cytometry for cell-bound extracellular HSP70:

  • Staining protocol:

    • Use non-permeabilizing conditions to detect only surface-bound HSP70

    • Compare with permeabilized samples to distinguish surface from intracellular pools

    • Recommended dilution: 1:1000 for FACS applications

  • Controls:

    • Include blocking steps to prevent non-specific binding

    • Use isotype controls at equivalent concentrations

• Western blotting of extracellular HSP70:

  • Sample concentration:

    • May require concentration of biological fluids or culture supernatants

    • TCA precipitation or ultrafiltration (10kDa cutoff) methods

  • Detection sensitivity:

    • Enhanced chemiluminescence for maximum sensitivity

    • Loading controls challenging for secreted proteins

• Extracellular vesicle (EV)-associated HSP70:

  • Isolation protocols:

    • Differential ultracentrifugation (100,000g)

    • Size exclusion chromatography

    • Immunocapture with EV markers

  • Analysis approaches:

    • Western blotting of EV lysates

    • Flow cytometry of captured EVs

    • Immunoelectron microscopy for visualization

These methodologies enable researchers to accurately detect and quantify extracellular HSP70, which has important implications for understanding its role in immune regulation and disease processes.

How can researchers optimize HSP70 antibody use for studying stress responses in different experimental models?

Optimizing HSP70 antibody use for stress response studies requires consideration of model-specific factors and experimental design:

• Cell culture stress models:

  • Heat shock protocol optimization:

    • Cell type-specific temperature and duration (typically 42-45°C for 20-60 minutes)

    • Recovery time course (0, 1, 3, 6, 24 hours post-stress)

    • Western blot detection using 1:1000 antibody dilution

  • Chemical stress inducers:

    • Heavy metals (cadmium, zinc)

    • Proteasome inhibitors (MG132, bortezomib)

    • Oxidative stressors (H₂O₂, paraquat)

  • Inhibitor studies:

    • KNK437 at 10-100 μM effectively inhibits HSP70 induction

    • Monitor dose-dependent effects on viability and HSP70 expression

• Animal model considerations:

  • Species cross-reactivity:

    • Many HSP70 antibodies cross-react with multiple species including mouse, rat, and human

    • Validate species-specific reactivity before extensive studies

  • Tissue-specific processing:

    • Different tissues require optimized extraction protocols

    • Include positive controls (e.g., heat-shocked samples)

• Stress response time course analysis:

  • Kinetic profiling:

    • HSP70 induction timing varies by stressor and cell/tissue type

    • Design time points to capture both early (1-3h) and late (6-24h) responses

    • Use multiple detection methods to confirm results

• Multiplexed detection approaches:

  • Co-staining protocols:

    • HSP70 with other stress markers (HSP90, HSF1)

    • HSP70 with cell death markers (cleaved caspase-3, PARP)

    • HSP70 with cell type-specific markers

  • Recommended staining dilutions:

    • 1:1000 for immunofluorescence applications

    • Optimize blocking to reduce background

• Quantitative analysis methods:

  • Western blot densitometry:

    • Normalize to loading controls (β-actin, GAPDH)

    • Use standard curves with recombinant protein for absolute quantification

  • Flow cytometry:

    • Mean fluorescence intensity measurement

    • Single-cell analysis of population heterogeneity

  • Immunofluorescence quantification:

    • Automated image analysis for consistency

    • Measure intensity, subcellular distribution, and co-localization

• Experimental data from stress models:

  Stress Type	HSP70 Induction Window	Detection Method	Antibody Dilution
  Heat shock (42°C)	1-24h, peak at 3-6h	Western blot	1:1000
  KNK437 inhibition	Dose-dependent reduction	Western blot	As used in HEL cells
  Oxidative stress	3-12h	Immunofluorescence	1:1000
  ER stress	6-24h	ELISA	Variable

These optimized approaches enable researchers to effectively study HSP70's critical role in stress responses across different experimental models, providing insights into both physiological and pathological processes.