HSPA12A Antibody

What is HSPA12A and what are its known biological functions?

HSPA12A (Heat shock 70 kDa protein 12A) is an atypical member of the HSP70 family. It functions as an adapter protein for SORL1 (but not SORT1), affecting SORL1 internalization kinetics and subcellular localization . Recent research has revealed several important physiological roles:

• In hepatocytes, HSPA12A inhibits macrophage chemotaxis and inflammation during liver ischemia-reperfusion (LI/R) injury by reducing glycolysis-generated lactate, which decreases HMGB1 lactylation and secretion .

• In the brain, HSPA12A controls cerebral lactate homeostasis, which is critical for maintaining mood stability .

• HSPA12A expression is tissue-specific, with highest levels observed in brain, kidney, and muscle tissues .

Understanding these functions is essential when designing experiments to study HSPA12A's role in various pathophysiological contexts.

Which applications are HSPA12A antibodies suitable for?

HSPA12A antibodies have been validated for multiple applications in molecular and cellular biology research:

• Western Blot (WB): For detecting denatured HSPA12A protein in tissue or cell lysates

• Immunohistochemistry (IHC): For visualization of HSPA12A in paraffin-embedded (IHC-P) or frozen tissue sections

• Immunofluorescence/Immunocytochemistry (IF/ICC): For subcellular localization studies in fixed and permeabilized cells

• Immunoprecipitation (IP): For isolation of HSPA12A protein complexes to study protein-protein interactions

The selection of the appropriate application depends on your research question. For instance, use WB for expression level quantification, IHC/IF for localization studies, and IP for protein interaction analyses.

How do I verify the specificity of HSPA12A antibodies?

Verification of antibody specificity is crucial for obtaining reliable results. Several approaches are recommended:

• Comparison with knockout/knockdown controls: Use HSPA12A knockout models or knockdown cells as negative controls. Research has utilized HSPA12A knockout models in hepatocytes that clearly demonstrate differences in protein detection compared to wild-type samples .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to verify that the signal disappears when the antibody is neutralized.

• Cross-validation with multiple antibodies: Use antibodies from different sources or those targeting different epitopes of HSPA12A. The search results indicate multiple validated antibodies are available from providers like LSBio, Boster Biological Technology, and Atlas Antibodies .

• Western blot molecular weight verification: HSPA12A has a calculated molecular weight of approximately 75 kDa, which should be confirmed on Western blots .

How does HSPA12A expression change during liver ischemia-reperfusion injury, and how can this be effectively measured?

HSPA12A expression in hepatocytes significantly decreases following liver ischemia-reperfusion (LI/R) injury. This downregulation appears to be more pronounced than changes in other heat shock proteins. The measurement of these changes requires careful experimental design:

• Transcript level analysis: qRT-PCR analysis reveals that among 29 Hsps measured, Hspa12a mRNA showed the greatest magnitude of decrease following LI/R compared to other downregulated Hsps .

• Protein level validation: Immunoblotting confirms decreased HSPA12A protein levels in both mouse LI/R livers and hepatocytes exposed to hypoxia/reoxygenation (H/R) conditions .

• Correlation with macrophage recruitment: The decrease in HSPA12A expression correlates with increased macrophage recruitment to the liver and to H/R-exposed hepatocytes, suggesting a functional relationship .

For accurate measurement, researchers should simultaneously assess both mRNA and protein levels while including appropriate time course analyses to capture the dynamic changes in expression during injury and recovery phases.

What mechanisms underlie HSPA12A's role in regulating HMGB1 lactylation and secretion?

HSPA12A regulates High-Mobility Group Box 1 (HMGB1) lactylation and secretion through a glycolysis-dependent mechanism. The pathway involves:

• Inhibition of glycolysis-generated lactate by HSPA12A overexpression

• Consequent decrease in HMGB1 lactylation in hepatocytes

• Reduced secretion of lactylated HMGB1 into the extracellular space

• Decreased macrophage chemotaxis and inflammatory activation

This mechanism was elucidated through gain- and loss-of-function studies showing that:

• HSPA12A overexpression attenuated LI/R-induced increases in serum HMGB1 levels in mice

• HSPA12A knockout in hepatocytes promoted H/R-induced HMGB1 lactylation and secretion

• The deleterious effects of HSPA12A knockout were reversed by HMGB1 knockdown

To investigate this mechanism, researchers should consider:

• Measuring lactate production in hepatocytes with varying HSPA12A expression levels

• Assessing HMGB1 lactylation status using mass spectrometry or lactylation-specific antibodies

• Quantifying intracellular HMGB1 translocation from nucleus to cytoplasm

• Monitoring exosomal HMGB1 secretion from hepatocytes

How can HSPA12A be effectively delivered to specific tissues for therapeutic applications?

Based on the emerging therapeutic potential of HSPA12A, particularly in liver and brain disorders, effective delivery methods have been developed:

For hippocampal delivery (relevant to mood disorders):

• Stereotaxic apparatus-guided administration of HSPA12A-adenovirus (3 × 10^10 PFU) to the hippocampus

• Precise injection at a rate of 200 nl/min followed by a 5-minute diffusion period

• Bilateral hippocampal injection for complete coverage

This approach has been successfully used to rescue phenotypes in Hspa12a^-/- mice .

For therapeutic development, researchers should consider:

• Vector selection (adenovirus vs. AAV) based on expression duration needs

• Promoter choice to ensure cell-type specificity

• Dose optimization to achieve physiological expression levels

• Controls using empty vectors to account for delivery-related effects

What are the optimal conditions for HSPA12A antibody-based immunocytochemistry?

For successful immunocytochemical detection of HSPA12A, the following protocol has been validated:

• Fixation: 4% paraformaldehyde fixation

• Permeabilization: 0.1% Triton X-100

• Primary antibody: Anti-HSPA12A at 1/250 dilution

• Secondary antibody: Fluorophore-conjugated anti-rabbit IgG (e.g., Alexa Fluor® 488) at 1/500 dilution

• Counter-staining: DAPI for nuclear visualization and cytoskeletal markers (e.g., α-tubulin) for structural context

This approach has been successfully used to detect cytoplasmic HSPA12A in cell lines such as U87-MG (human glioblastoma) .

For optimal results, consider:

• Testing antibody dilutions to determine the optimal signal-to-noise ratio

• Including appropriate negative controls (primary antibody omission, isotype controls)

• Using HSPA12A-knockdown cells as specificity controls

• Confirming subcellular localization with organelle-specific markers

What experimental models are most suitable for studying HSPA12A function in different contexts?

Various experimental models have been developed to study HSPA12A function:

For liver ischemia-reperfusion studies:

• In vivo mouse LI/R model with hepatocyte-specific HSPA12A overexpression (h-Ki mice)

• Primary hepatocyte cultures exposed to hypoxia/reoxygenation (H/R)

• HSPA12A knockout hepatocytes to evaluate loss-of-function effects

• Co-culture systems of hepatocytes and macrophages to study cell-cell interactions

For neurological and mood disorder studies:

• Hspa12a^-/- knockout mice with targeted hippocampal HSPA12A restoration via adenoviral delivery

• Wild-type controls and empty vector controls

When selecting an appropriate model, researchers should consider:

• The specific biological context being investigated (liver, brain, other tissues)

• Whether gain-of-function or loss-of-function approaches are more informative

• The need for cell-type specific manipulation versus systemic effects

• Acute versus chronic experimental timelines

How can I quantitatively analyze HSPA12A-dependent effects on inflammatory responses?

To quantitatively assess HSPA12A's impact on inflammatory responses, several validated approaches can be employed:

• Hepatic inflammatory mediator expression:

  • qRT-PCR analysis of inflammatory cytokines (TNFα, IL6) in liver tissue samples

  • Comparison between wild-type and HSPA12A-overexpressing mice following LI/R

• Macrophage chemotaxis quantification:

  • Assessment of macrophage recruitment to liver tissue in vivo using immunohistochemistry or flow cytometry

  • In vitro quantification of macrophage migration toward conditioned medium from hepatocytes with varying HSPA12A expression

• Hepatocyte injury markers:

  • Measurement of ALT and AST release from hepatocytes with different HSPA12A expression levels following H/R exposure

  • Assessment of further injury following co-culture with activated macrophages

• HMGB1 secretion analysis:

  • ELISA for HMGB1 in serum samples from experimental animals

  • Immunoblotting of cytosolic HMGB1 fractions to assess translocation

  • Quantification of HMGB1 in hepatocyte culture medium following H/R

For robust analysis, researchers should:

• Include appropriate time points to capture the dynamics of inflammatory responses

• Use multiple complementary methodologies (e.g., gene expression, protein levels, functional assays)

• Implement appropriate statistical analyses to account for biological variability

• Include both in vitro and in vivo validation when possible

What are common issues with HSPA12A Western blotting and how can they be resolved?

When performing Western blot analysis with HSPA12A antibodies, researchers may encounter several challenges:

• Multiple bands/non-specific binding:

  • Issue: Detection of bands at molecular weights other than the expected 75 kDa

  • Resolution: Optimize blocking conditions (5% non-fat milk or BSA), antibody dilution, and washing steps

  • Validation: Use HSPA12A knockout samples as negative controls to identify specific bands

• Weak signal:

  • Issue: Insufficient detection of HSPA12A, particularly in tissues with lower expression

  • Resolution: Increase protein loading (up to 50 μg), optimize antibody concentration, or use enhanced chemiluminescence detection systems

  • Enhancement: Consider using signal amplification systems or longer exposure times while monitoring background

• Tissue-specific expression variation:

  • Issue: HSPA12A levels vary significantly across tissues, with highest expression in brain, kidney, and muscle

  • Resolution: Adjust protein loading and antibody dilutions based on expected expression levels in different tissues

  • Control: Include positive control samples from high-expressing tissues

• Standardization across experiments:

  • Issue: Variability in HSPA12A detection between experimental batches

  • Resolution: Include consistent positive controls in each experiment and normalize to housekeeping proteins

  • Documentation: Maintain detailed records of antibody lot numbers, detection reagents, and exposure conditions

How do I select the most appropriate HSPA12A antibody for my specific research application?

Selection of the optimal HSPA12A antibody depends on several factors:

• Application specificity:

  • For Western blotting: Select antibodies specifically validated for WB (numerous options available from providers like Affinity Biosciences, LSBio, and Abcam)

  • For immunohistochemistry: Choose antibodies validated for IHC-P or IHC-F depending on your sample preparation

  • For multiple applications: Consider antibodies validated across several techniques (e.g., Boster Biological Technology M13632 validated for WB, ICC, IP, and IHC)

• Species reactivity:

  • Ensure the antibody reacts with your species of interest (human, mouse, rat)

  • For less common species, consider antibodies with predicted cross-reactivity based on sequence homology (e.g., pig, bovine, horse)

• Antibody format:

  • Monoclonal antibodies (e.g., Abcam EPR16763) for consistent results across experiments

  • Polyclonal antibodies for potentially higher sensitivity in applications like IHC

• Validation evidence:

  • Prioritize antibodies with published validation data or references

  • Consider those with enhanced validation or supportive data in repositories like Antibodypedia

• Epitope considerations:

  • Select antibodies targeting conserved regions when working across species

  • Choose different epitope-targeting antibodies when seeking confirmation through multiple antibodies