HSPA8 Antibody

What is HSPA8 and why is it important in cellular research?

HSPA8 (Heat Shock Protein Family A Member 8), also known as HSC70 or HSP73, is a constitutively expressed molecular chaperone that plays critical roles in multiple cellular processes. Unlike inducible heat shock proteins, HSPA8 is expressed under normal conditions and is crucial for:

• Protein quality control, ensuring proper folding and refolding of selected proteins

• Regulation of chaperone-mediated autophagy (CMA) as a detector of substrates

• Anti-bacterial autophagy through interaction with proteins like RHOB and BECN1

• Prevention of necroptosis by acting as an amyloidase that inhibits RHIM-amyloid formation

• Multiple immune system regulatory functions

The importance of HSPA8 in research stems from its involvement in fundamental cellular processes and its dysregulation in various disease states, including autoimmune disorders and bacterial infections.

What types of HSPA8 antibodies are available for research applications?

Several types of HSPA8 antibodies are available for research applications, varying in their target regions, host species, and recommended applications:

When selecting an HSPA8 antibody, researchers should consider:

• Target region of interest (N-terminal vs. C-terminal epitopes)

• Required applications (some antibodies perform better in specific applications)

• Species cross-reactivity (particularly important for comparative studies)

What are the optimal conditions for using HSPA8 antibodies in Western blotting?

For optimal Western blot results with HSPA8 antibodies, follow these methodological guidelines:

Sample Preparation:

• Use appropriate lysis buffers containing protease inhibitors

• Load 25-35 μg of total protein per lane for cell lines (as demonstrated in validated protocols)

Running Conditions:

• Use reducing conditions with standard SDS-PAGE (typically 10-12% gels)

• Include positive control samples (validated cell lines include HeLa, A431, NIH/3T3)

Antibody Dilutions:

• Primary antibody: 1:1000 dilution for most commercial HSPA8 antibodies

• Anti-HSPA8 N-terminal antibody: 1:1000 dilution

• Secondary antibody: typically 1:5000 dilution (HRP-conjugated anti-rabbit or anti-mouse IgG)

Detection:

• Expected molecular weight: 70-72 kDa

• Allow sufficient exposure time (HSPA8 is generally abundant but may vary by cell type)

• For reproducible results, standardize protein loading using housekeeping protein controls

Note that HSPA8 is highly conserved across species, allowing most antibodies to detect human, mouse, and rat orthologs with similar efficiency .

How should I optimize HSPA8 immunofluorescence staining protocols?

For optimal immunofluorescence results when detecting HSPA8:

Cell Preparation:

• Use appropriate fixation: 4% paraformaldehyde (10-15 minutes) works well for most applications

• Permeabilization: 0.1-0.2% Triton X-100 for 5-10 minutes at room temperature

Staining Protocol:

• Blocking: 1-5% BSA or normal serum from secondary antibody host species (1 hour at room temperature)

• Primary antibody concentration: 10 μg/mL for the mouse monoclonal anti-HSPA8/HSC71 antibody

• Incubation time: 3 hours at room temperature or overnight at 4°C

• Secondary antibody: fluorophore-conjugated anti-mouse or anti-rabbit IgG (depending on primary)

Expected Localization Pattern:

• Primarily cytoplasmic distribution

• Nuclear localization, especially in nucleoli

• Potential punctate structures under stress conditions or specific experimental treatments

Co-staining Recommendations:

• DAPI for nuclear counterstaining

• Phalloidin for actin filament visualization to provide cellular context

• For autophagy studies, consider co-staining with LC3 or LAMP2A

Validated examples show clear nuclear and nucleolar localization in HeLa cells when using mouse anti-HSPA8/HSC71 at 10 μg/mL with NorthernLights 557-conjugated secondary antibody .

How can I use HSPA8 antibodies to study its role in anti-bacterial autophagy?

HSPA8 plays a crucial role in anti-bacterial autophagy through its interactions with RHOB and BECN1. To effectively study this function:

Experimental Design:

• Bacterial Infection Models:

  • Select appropriate bacterial strains: Validated models include Salmonella typhi LT2 in SW480 cells and uropathogenic Escherichia coli CFT073 in 5637 cells

  • Establish consistent infection protocols (MOI, infection time)

• HSPA8 Manipulation Approaches:

  • siRNA knockdown of HSPA8 (validated in both cell culture and mouse models)

  • Overexpression of wild-type HSPA8 or domain-specific mutants (particularly IDR domain mutants)

  • Pharmacological modulators of HSPA8 function

• Readout Assays:

  • Quantification of intracellular bacteria (CFU assays, immunofluorescence)

  • LC3-decoration of bacteria (co-localization studies)

  • Autophagy marker analysis (LC3-II, BECN1, SQSTM1 levels by Western blot)

Methodological Example:
Research has shown that HSPA8 knockdown significantly impairs bacterial clearance. This can be assessed by:

• Transfecting cells with HSPA8-specific siRNAs

• Infecting with bacteria (e.g., S. typhi LT2 or UPEC CFT073)

• Measuring bacterial load at different time points

• Comparing with control siRNA-treated cells

Additionally, you can study the mechanism by examining:

• HSPA8-RHOB-BECN1 complex formation using co-immunoprecipitation

• Domain-specific interactions using truncated constructs

• LLPS (liquid-liquid phase separation) properties using fluorescently-tagged proteins

Research has demonstrated that HSPA8, through its IDR domain (residues 243-283), promotes LLPS to concentrate RHOB and BECN1, enhancing their interaction and stabilization .

What approaches should I use to study HSPA8's role in chaperone-mediated autophagy (CMA)?

Studying HSPA8's role in CMA requires specific methodological approaches:

Experimental System Selection:

• Cell lines with high CMA activity (e.g., fibroblasts, hepatocytes)

• Tissues with notable CMA dependency (liver, brain, immune cells)

CMA Substrate Tracking:

• Use established CMA substrates containing KFERQ-like motifs (e.g., GAPDH, RNase A)

• Generate fluorescently-tagged CMA substrate reporters

• Employ pulse-chase experiments to track substrate degradation

HSPA8-LAMP2A Interaction Studies:

• Co-immunoprecipitation assays to assess HSPA8-LAMP2A binding

• Proximity ligation assays for in situ visualization of interactions

• FRET/BRET approaches for real-time interaction monitoring

CMA Activity Assessment:

• Lysosomal isolation and in vitro uptake assays

• Selective degradation of known CMA substrates

• LAMP2A multimerization analysis

Specific Methodology Example:
To determine if a protein is degraded via CMA involving HSPA8:

• Generate cells with HSPA8 knockdown or overexpression

• Measure half-life of candidate CMA substrates in presence/absence of lysosomal inhibitors

• Perform competition assays with known KFERQ-containing peptides

• Assess direct binding of the substrate to HSPA8 using purified proteins

• Evaluate LAMP2A dependency through selective LAMP2A knockdown

Remember that HSPA8 recognizes and delivers KFERQ-motif containing proteins with the help of co-chaperones for degradation via macroautophagy, CMA, or endosomal microautophagy (eMI) pathways .

Why might I observe inconsistent HSPA8 staining patterns across different cell types?

Inconsistent HSPA8 staining patterns across different cell types can result from several factors:

Biological Factors:

• Differential Expression Levels:

  • HSPA8 expression varies naturally between cell types and tissues

  • In some immune disorders, expression is altered (e.g., elevated in MRL/lpr lupus-prone mice B cells, T cells, and CD11b+Gr-1+ granulocytes/macrophages)

• Subcellular Localization Differences:

  • HSPA8 shuttles between cellular compartments depending on cell state

  • Stress conditions can alter localization patterns

  • Interaction with different partners affects distribution

• Post-translational Modifications:

  • Phosphorylation state affects localization and function

  • Ubiquitination can alter stability and detection

Technical Considerations:

• Fixation Method Impact:

  • Paraformaldehyde vs. methanol fixation reveals different epitopes

  • Overfixation can mask epitopes, particularly affecting N-terminal antibodies

• Antibody Specificity:

  • N-terminal antibodies (aa 82-110) may detect different conformations than C-terminal antibodies (aa 534-615)

  • Some antibodies may recognize specific functional states

• Protocol Optimization Requirements:

  • Cell-type specific permeabilization conditions

  • Blocking protocol adjustments

  • Primary antibody concentration titration

Recommendations for Standardization:

• Include positive control cell lines (HeLa cells show consistent nuclear/nucleolar staining)

• Optimize fixation time for each cell type

• Consider dual-antibody approaches (N-terminal and C-terminal antibodies) for complete picture

• When comparing cell types, process and image them under identical conditions

How can I distinguish between HSPA8 and other HSP70 family members in my experimental data?

Distinguishing HSPA8 from other HSP70 family members is crucial for accurate data interpretation due to their high sequence homology. Here's a methodological approach:

Antibody Selection Strategy:

• Epitope Targeting:

  • Select antibodies raised against unique regions (typically C-terminal domains)

  • Verify epitope specificity against sequence alignments of HSP70 family members

  • Examine published validation data showing specificity testing

• Validation Using Knockout/Knockdown Controls:

  • Include HSPA8 knockout or knockdown samples as negative controls

  • Test antibody reactivity against recombinant HSPA8 and other HSP70 proteins

Experimental Approaches:

• Western Blot Discrimination:

  • Use gradient gels (8-12%) to resolve subtle size differences

  • Include positive controls for other HSP70 family members

  • Consider 2D gel electrophoresis to separate based on both MW and pI

• Immunoprecipitation Specificity:

  • Perform IP-Mass Spectrometry to confirm identity

  • Use stringent washing conditions to minimize cross-reactivity

• Expression Pattern Analysis:

  • HSPA8 is constitutively expressed, while HSPA1A/B are stress-inducible

  • Examine expression under normal vs. stress conditions

Mass Spectrometry Approach:
For definitive identification, use MS-based methods:

• Immunoprecipitate with HSPA8 antibody

• Digest and analyze by LC-MS/MS

• Identify unique peptides specific to HSPA8 vs. other family members

Documented Distinction Methods:
Research has demonstrated effective discrimination through:

• Using antibodies targeting the C-terminal region (aa 534-615)

• Conducting parallel knockdown experiments for verification

• Employing isoform-specific PCR to correlate protein with mRNA expression

How can HSPA8 antibodies be utilized to investigate liquid-liquid phase separation (LLPS) in autophagy regulation?

LLPS is an emerging mechanism in cellular biology, and HSPA8 has recently been identified as a key player in LLPS-mediated autophagy regulation. Here's how to investigate this phenomenon:

Experimental Approaches:

• In Vitro Phase Separation Assays:

  • Purify recombinant HSPA8 protein (full-length and domain variants)

  • Monitor droplet formation using microscopy under various conditions (protein concentration, salt, pH)

  • Use fluorescently-labeled HSPA8 to visualize droplet dynamics

• Cellular LLPS Visualization:

  • Express fluorescently-tagged HSPA8 constructs (GFP-HSPA8)

  • Employ live-cell imaging to monitor droplet formation

  • Test HSPA8 mutants (particularly IDR domain mutants) for LLPS capability

• HSPA8-Mediated Concentration of Autophagy Factors:

  • Co-express tagged RHOB and BECN1 with HSPA8

  • Quantify co-localization in liquid-like condensates

  • Assess functional consequences through autophagy assays

Methodological Details:
Research has demonstrated that HSPA8 contains predicted intrinsically disordered regions (IDRs) and drives LLPS to concentrate RHOB and BECN1 into HSPA8-formed liquid-phase droplets . To study this:

• LLPS Demonstration:

  • Express GFP-HSPA8 in HEK293 cells and observe puncta formation

  • Confirm liquid properties using 1,6-hexanediol treatment (dissolves liquid condensates)

  • Perform FRAP (Fluorescence Recovery After Photobleaching) to verify liquid-like behavior

• Functional Impact Assessment:

  • Generate HSPA8 constructs lacking the IDR domain (HSPA8Δ243-283)

  • Compare wild-type and mutant HSPA8 for:

    • Ability to form condensates

    • Capacity to concentrate RHOB and BECN1

    • Effect on bacterial clearance

• Client Protein Concentration:

  • Quantify BECN1 and RHOB recruitment to HSPA8 droplets

  • Measure interaction strength using FRET or biochemical assays

  • Correlate condensate formation with autophagy induction

Research has shown that HSPA8 lacking the IDR domain failed to form puncta and was unable to effectively concentrate RHOB and BECN1, resulting in impaired bacterial clearance .

What are the best approaches to study HSPA8's role in immune disorders using specific antibodies?

HSPA8 has been implicated in various immune disorders, particularly autoimmune diseases like systemic lupus erythematosus. Here are methodological approaches using HSPA8 antibodies:

Cellular Distribution Analysis:

• Immune Cell Phenotyping:

  • Use flow cytometry with HSPA8 antibodies to quantify expression across immune cell subsets

  • Compare HSPA8 levels between healthy controls and disease samples

  • Correlate with disease activity markers

• Tissue-Specific Expression:

  • Perform IHC on tissue sections from affected organs

  • Quantify HSPA8 expression in specific cell types within the tissue

  • Co-stain with cell type markers and disease-associated proteins

Functional Studies:

• Antigen Presentation Role:

  • Assess HSPA8 involvement in MHC class II presentation pathway

  • Study co-localization with processed antigens in antigen-presenting cells

  • Analyze effect of HSPA8 modulation on T cell activation

• Autophagy Regulation in Immune Cells:

  • Measure CMA activity in different immune cell populations

  • Correlate with HSPA8 expression and localization

  • Determine impact on immune cell function and survival

Disease-Specific Applications:
Research has shown that in lupus-prone mice, HSPA8 expression is elevated in multiple immune cell types . To investigate this:

• Comparative Expression Analysis:

  • Use flow cytometry with HSPA8 antibodies to quantify surface and intracellular expression

  • Compare B cells, T cells, activated T cells, and CD11b+Gr-1+ granulocytes/macrophages

  • Correlate with mRNA expression levels

• Mechanistic Investigation:

  • Assess HSPA8's role in autoantigen presentation

  • Determine if HSPA8 modulation affects autoantibody production

  • Study HSPA8-client protein interactions in disease states

• Therapeutic Target Validation:

  • Test HSPA8-targeting compounds in disease models

  • Monitor immune parameters and disease progression

  • Identify specific pathways affected by HSPA8 modulation

These approaches can provide valuable insights into HSPA8's role in immune disorders and potentially identify new therapeutic strategies targeting this chaperone protein .

Human HSPA8 Antibody Research: Current Status and Future Directions

This FAQ collection represents the current state of knowledge regarding HSPA8 antibodies and their applications in research. As scientific understanding continues to evolve, researchers are encouraged to:

• Validate antibodies in their specific experimental systems

• Consider the growing role of HSPA8 in cellular phase transitions and compartmentalization

• Explore the therapeutic potential of targeting HSPA8 in infectious and autoimmune diseases

• Employ complementary approaches (genetic, biochemical, imaging) for comprehensive insights into HSPA8 function