HSP70 Antibody

What is HSP70 and why is it significant in research?

HSP70 belongs to a family of heat shock proteins that function as molecular chaperones. They play crucial roles in protein folding, oligomerization, and intracellular transport. The universal ability of HSP70s to undergo cycles of binding to and release from hydrophobic stretches of partially unfolded proteins determines their role in a great variety of vital intracellular functions . HSP70 is particularly interesting to researchers because it can be found both intracellularly and on cell membranes, with membrane-bound HSP70 being uniquely present on cancer cells but not normal cells, suggesting a conformational change in the lower pH environment characteristic of cancer cells . This differential expression makes HSP70 a valuable target for cancer research and potential therapeutic applications.

What applications are HSP70 antibodies commonly used for?

HSP70 antibodies are versatile research tools with multiple applications:

• Western Blotting: Detects HSP70 protein expression levels in cell or tissue lysates, typically appearing as a band at approximately 70-72 kDa .

• Immunohistochemistry (IHC): Visualizes HSP70 distribution in tissue sections, particularly useful for comparing expression between normal and diseased tissues .

• Immunofluorescence (IF): Localizes HSP70 within cells, allowing discrimination between membrane-bound and intracellular HSP70 .

• Flow Cytometry (FACS): Quantifies HSP70 expression on cell surfaces or intracellularly across cell populations .

• ELISA: Measures HSP70 levels or anti-HSP70 antibodies in biological fluids .

• Immunoelectron Microscopy (IEM): Provides ultra-structural localization of HSP70 .

• Antibody Array and BioImaging: Offers high-throughput analysis and visualization of HSP70 expression patterns .

How do I select the appropriate HSP70 antibody for my research?

Selecting the right HSP70 antibody depends on multiple factors:

• Specificity: Determine whether you need an antibody that recognizes inducible HSP70 specifically or one that might cross-react with constitutive HSC70. For example, some antibodies like clone C92F3A-5 specifically detect HSP70 (~70 kDa) without cross-reacting with HSC70 (HSP73) .

• Species reactivity: Confirm the antibody recognizes HSP70 in your species of interest. Some antibodies offer broad cross-reactivity across species (human, mouse, rat, etc.) .

• Application compatibility: Verify the antibody has been validated for your specific application. For instance, AF1663 has been validated for Western blot in human, mouse, and rat samples .

• Clone type: Consider whether a monoclonal (greater specificity) or polyclonal (potentially greater sensitivity) antibody better suits your needs.

• Membrane vs. intracellular HSP70: For cancer research, special consideration should be given to antibodies that can distinguish between membrane-bound and intracellular HSP70, such as clone 1H11 (SMC-249) .

How can I validate the specificity of HSP70 antibodies?

Proper validation ensures experimental reliability:

• Positive and negative controls: Include cell lines known to express high levels of HSP70 (e.g., heat-shocked Jurkat cells) as positive controls, and appropriate negative controls .

• Multiple detection methods: Confirm findings using alternative techniques (e.g., if using Western blot, verify with immunofluorescence).

• Heat shock induction: Compare HSP70 levels in heat-shocked versus non-shocked cells, as inducible HSP70 increases significantly after heat stress .

• Molecular weight verification: Confirm the detected band appears at the expected molecular weight (~70-72 kDa) .

• Knockout/knockdown validation: If possible, use HSP70 knockout or knockdown cells to confirm antibody specificity.

• Peptide competition: Pre-incubation of the antibody with the immunizing peptide should eliminate specific staining.

What protocols are recommended for detecting membrane-bound versus intracellular HSP70?

Distinguishing between membrane-bound and intracellular HSP70 is critical, particularly in cancer research:

Immunofluorescence protocol for membrane HSP70:

• Fix cells with 4% formaldehyde (minimal permeabilization).

• Use an antibody specific for membrane-bound HSP70, such as clone 1H11 (SMC-249) at 1:100 dilution.

• Counter-stain with membrane markers (e.g., wheat germ agglutinin) and nuclear stains (DAPI).

• Analyze using confocal microscopy to distinguish membrane from intracellular localization .

Flow cytometry for membrane HSP70:

• Use live cells without fixation or permeabilization.

• Incubate with anti-HSP70 antibody (e.g., clone 1H11) at 4°C.

• Add propidium iodide to exclude dead cells.

• This approach has successfully shown that HSP70 antibodies bind to the cell surface of tumor cells but not non-tumor cells .

How can I optimize Western blot protocols for HSP70 detection?

For optimal Western blot results with HSP70 antibodies:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors.

  • Heat samples at 95°C for 5 minutes in reducing buffer.

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal separation.

  • Include positive controls such as heat-shocked cell lysates.

• Transfer and blocking:

  • Transfer proteins to PVDF membrane.

  • Block with 5% non-fat dry milk or BSA in TBST.

• Antibody incubation:

  • Primary antibody: Use at optimized concentration (e.g., 0.1-0.5 μg/mL for AF1663).

  • Secondary antibody: HRP-conjugated anti-species IgG.

• Detection:

  • Use enhanced chemiluminescence detection systems.

  • Expect HSP70 bands at approximately 70-72 kDa .

• Special considerations:

  • For comparing HSP70 levels between samples, consider using GAPDH as an internal control .

  • When comparing normal vs. stressed conditions, heat shock treatment can serve as a positive control for inducible HSP70 upregulation .

What are the best practices for flow cytometry analysis of HSP70?

Flow cytometry allows quantitative assessment of HSP70 expression:

• Cell surface HSP70 detection:

  • Use live, unfixed cells to prevent permeabilization.

  • Incubate cells with HSP70 antibody (e.g., clone 1H11) at 4°C for 40-90 minutes.

  • Use propidium iodide (2.5 μg/ml) for 5 minutes at 4°C to exclude dead cells.

  • This approach has successfully demonstrated HSP70 expression across multiple cancer cell lines (Jurkat, U937, MCF7, HT29) .

• Intracellular HSP70 detection:

  • Fix cells with formaldehyde or methanol.

  • Permeabilize with saponin or Triton X-100.

  • Block with appropriate serum.

  • Incubate with anti-HSP70 antibody followed by fluorescent secondary antibody.

• Controls:

  • Include appropriate isotype controls.

  • Use known positive cell lines (e.g., heat-shocked cells).

  • Compare tumor cell lines with non-tumor cells (e.g., HCT116 vs. HK2 cells) .

How do I design experiments to study HSP70's role in cancer?

HSP70's differential expression in cancer makes it a valuable research target:

• Comparative expression studies:

  • Use immunofluorescence and flow cytometry to compare HSP70 localization and levels between cancer and normal cell lines.

  • Research indicates HSP70 is localized on the cell surface of cancer cells but not normal cells .

• Functional studies:

  • Investigate how membrane-embedded HSP70 increases cancer cell stability and protects tumors from environmental stress .

  • Design experiments to target membrane HSP70 with specific antibodies and measure effects on cancer cell survival.

• Mechanistic investigations:

  • Study how the pH environment affects HSP70 conformation and membrane localization in cancer cells.

  • Investigate downstream signaling pathways activated by HSP70 in cancer cells.

• Therapeutic potential:

  • Design experiments to test whether targeting membrane HSP70 with antibodies can selectively affect cancer cells while sparing normal cells.

  • Measure changes in cell viability, apoptosis, and stress response.

How can HSP70 antibodies be used in therapeutic applications?

Emerging research highlights HSP70's therapeutic potential:

• Immunotherapy approaches:

  • HSP70 immunization in mouse models has shown promising results in psoriasis-like skin inflammation, resulting in decreased clinical and histological disease severity .

  • Anti-HSP70 antibody treatment has demonstrated lowered disease activity in psoriasis models .

• Immune modulation mechanisms:

  • HSP70 immunization appears to work by expanding T cells in favor of regulatory subtypes (CD4+FoxP3+/CD4+CD25+ cells) .

  • Anti-HSP70 antibody treatment is associated with down-regulation of pro-inflammatory Th17 cells and an increase in the CD4+FoxP3+:Th17 ratio .

• Experimental design considerations:

  • When testing anti-HSP70 therapies, measure both clinical parameters and immunophenotypic changes.

  • Consider assessing T cell subpopulations (Tregs, Th17) to understand the mechanism of action.

  • Monitor both local (skin) and systemic (splenic, blood) immune changes to get a complete picture of therapeutic effects .

What role does HSP70 play in autoimmune disorders?

HSP70's involvement in autoimmunity offers research opportunities:

• Autoantibodies to HSP70:

  • Anti-HSP70 autoantibodies can be detected in biological fluids like saliva and serum, even in healthy individuals .

  • Changes in these autoantibody levels may correlate with certain disease states.

• Experimental approaches:

  • ELISA protocols can detect anti-HSP70 antibodies in biological samples. Plates can be coated with HSP70 (0.5 μg/ml) in bicarbonate buffer, blocked with BSA, and incubated with diluted serum/saliva samples .

  • Measure autoantibody titers in patient samples versus healthy controls across different autoimmune conditions.

• Immunomodulatory effects:

  • HSP70 has direct stimulating action on regulatory T cells, which can be studied in cell culture experiments .

  • Research suggests HSP70 may have anti-proliferative effects on keratinocytes, relevant to skin disorders like psoriasis .

What methodology should be used to study HSP70's effects on immune cell populations?

Detailed protocols for studying HSP70's immunomodulatory effects:

• Flow cytometry for immune cell characterization:

  • For regulatory T cells: Measure CD4+FoxP3+ (spleen) and CD4+CD25+ (blood) frequencies.

  • For Th17 cells: Detect CD4+IL-17+ cells.

  • Calculate CD4+FoxP3+:Th17 ratio as an indicator of immune balance .

• In vivo experimental design:

  • HSP70 immunization: Administer recombinant HSP70 and monitor immune responses.

  • Anti-HSP70 treatment: Use well-characterized antibodies against HSP70 and evaluate:

    • Disease parameters (e.g., skin inflammation in psoriasis models)

    • T cell subpopulations

    • Pro-inflammatory vs. anti-inflammatory balance .

• In vitro functional assays:

  • Regulatory T cell stimulation assays with purified HSP70.

  • Keratinocyte proliferation assays in the presence of HSP70 or anti-HSP70 antibodies.

  • Cytokine production measurement from immune cells exposed to HSP70 .

How can I design experiments to investigate HSP70's role in stress response signaling?

HSP70's core function in stress response merits detailed investigation:

• Stress induction protocols:

  • Heat shock: Expose cells to elevated temperatures (42-45°C) for defined periods.

  • Oxidative stress: Treat with H₂O₂ or other oxidants.

  • Chemical stress: Apply proteasome inhibitors or other stress-inducing compounds.

• HSP70 expression analysis:

  • Time-course experiments to track HSP70 induction (6h, 24h, etc.).

  • Western blot to quantify protein levels using appropriate loading controls like GAPDH .

  • qRT-PCR to measure mRNA expression changes.

• Signaling pathway analysis:

  • Investigate relationship between HSP70 and MyD88-dependent pathways.

  • Consider using pathway inhibitors (e.g., Pepinh-MyD) to determine specificity.

  • Compare wild-type with knockout models (e.g., MyD88 KO mice) to establish pathway dependence .

• Functional readouts:

  • Cell viability/apoptosis assays under stress conditions.

  • Protein aggregation and refolding assays.

  • Immune activation measurements (e.g., antibody production following immunization) .

What are common pitfalls when working with HSP70 antibodies and how can they be addressed?

Addressing technical challenges improves experimental outcomes:

• Cross-reactivity issues:

  • Problem: Unintended detection of HSC70 (constitutive form).

  • Solution: Use antibodies specifically validated for HSP70 vs. HSC70 discrimination, such as clone C92F3A-5 which does not cross-react with HSC70 .

• Background in immunostaining:

  • Problem: Non-specific binding creates high background.

  • Solution: Optimize blocking (1% BSA recommended), use appropriate antibody dilutions (e.g., 1:100 for immunofluorescence with SMC-249), and include proper negative controls .

• Inconsistent Western blot results:

  • Problem: Variable band intensity or unexpected bands.

  • Solution: Ensure consistent lysate preparation, optimize antibody concentration (0.1-0.5 μg/mL recommended for AF1663), and use reducing conditions with appropriate buffer systems .

• Flow cytometry challenges:

  • Problem: Difficulty distinguishing membrane from intracellular HSP70.

  • Solution: For membrane-only detection, use unfixed cells at 4°C with short incubation times (40-90 minutes); for total HSP70, use fixed and permeabilized cells .

How should HSP70 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling ensures antibody performance:

• Storage conditions:

  • Temperature: Store antibodies at -20°C for long-term storage or at 4°C for short-term use.

  • Format: Small pack sizes are typically supplied either lyophilized or as 0.2 μm filtered solutions in PBS .

  • Avoid repeated freeze-thaw cycles by aliquoting antibodies upon first thaw.

• Working dilutions:

  • Western blot: Typically 0.1-0.5 μg/mL

  • Immunofluorescence: 1:100 dilution has been validated for certain antibodies

  • Flow cytometry: 20 μg/ml for 40 minutes at 4°C has shown good results

• Reconstitution of lyophilized antibodies:

  • Use sterile techniques

  • Reconstitute in recommended buffer (typically PBS)

  • Allow complete dissolution before use

• Quality control measures:

  • Include positive controls in each experiment

  • Periodically validate antibody performance

  • Monitor for changes in specificity or sensitivity over time

Research Application Table

The following table summarizes key HSP70 antibody applications and recommended parameters: