HSPA12A Antibody, HRP conjugated

What is HSPA12A and what biological functions does it serve?

HSPA12A (Heat shock 70 kDa protein 12A) is a member of the heat shock protein family A (HSPA), which has been identified as a novel adaptor protein that selectively binds to SorLA among Vps10p-D receptors. The interaction between HSPA12A and SorLA occurs in an ADP/ATP-dependent manner, with HSPA12A binding to specific acidic residues in the cytoplasmic domain (cd) of SorLA. This binding affects both the endocytic speed and subcellular localization of SorLA, essentially delaying SorLA internalization . Unlike typical heat shock proteins that primarily function as chaperones, HSPA12A appears to have specialized in protein trafficking regulation. The discovery of HSPA12A as a SorLA-specific interaction partner provides novel insight into the molecular mechanism of SorLA and emphasizes the role of heat shock proteins in neurodegenerative diseases .

Where is HSPA12A predominantly expressed in tissues?

HSPA12A exhibits expression in multiple tissues with varying intensities. Immunohistochemical analysis has confirmed expression in human cerebral cortex tissue, where it shows cytoplasmic staining patterns . Western blot analyses have also detected HSPA12A in human fetal kidney tissue, mouse spleen tissue, and rat heart tissue, all revealing the protein at its predicted molecular weight of 75 kDa . Additionally, the protein has been detected in fetal brain tissue through immunoprecipitation techniques . In primary cortical astrocytes, HSPA12A demonstrates dual localization in both soluble (cytoplasmic) and insoluble (nuclear) fractions, a feature shared with several members of the HSP70 family . Microscopically, HSPA12A appears associated with perinuclear compartments and vesicular-like structures throughout the cytoplasm, with partial co-localization with SorLA particularly in perinuclear compartments/vesicles .

How does HSPA12A differ from other heat shock proteins?

HSPA12A represents a distinct subtype within the heat shock protein family A. Unlike canonical heat shock proteins that function primarily as molecular chaperones facilitating protein folding and preventing aggregation, HSPA12A has evolved specialized functions in protein trafficking regulation. Research has demonstrated that HSPA12A specifically targets the cytoplasmic domain of SorLA but not Sortilin (SORT1), indicating a high degree of substrate specificity not typically observed in other heat shock proteins . Furthermore, HSPA12A exhibits structural differences from its close relative HSPA12B; when testing the corresponding C-terminal fragment from HSPA12B against the SorLA cytoplasmic domain, no interaction was observed, highlighting functional divergence within the HSPA12 subfamily . Additionally, HSPA12A retains the characteristic nucleotide-dependent conformational changes observed in the HSP70 family, with ADP-bound forms demonstrating elevated substrate binding activity compared to ATP-bound forms .

What detection methods are most effective when using HSPA12A antibodies?

When working with HSPA12A antibodies, multiple detection methods have proven effective for different experimental objectives. Western blotting provides reliable detection using HSPA12A-specific antibodies at dilutions of 1:1000, with observed bands consistently appearing at the predicted molecular weight of 75 kDa across multiple tissue types . For immunohistochemistry on paraffin-embedded tissues, HSPA12A antibodies perform optimally at 1:100 dilution following heat-mediated antigen retrieval with Tris/EDTA buffer at pH 9.0 . Immunofluorescence microscopy can effectively visualize both nuclear and cytoplasmic HSPA12A localization, particularly in primary cortical astrocytes where it appears in perinuclear compartments and vesicular structures . For co-localization studies with SorLA, dual immunofluorescence labeling combined with confocal microscopy provides the necessary resolution to observe partial co-localization in specific subcellular compartments . For protein-protein interaction studies, both Yeast-Two-Hybrid (Y2H) screening and GST pull-down assays have successfully demonstrated the interaction between HSPA12A and SorLA, with the latter particularly useful for examining nucleotide-dependent binding dynamics .

What are the critical parameters for optimizing Western blot detection of HSPA12A?

Optimizing Western blot detection of HSPA12A requires careful attention to several critical parameters. First, protein extraction conditions significantly impact results—for comprehensive analysis, separate extraction of both Triton X-100 soluble and insoluble fractions is recommended, as HSPA12A distributes between cytoplasmic and nuclear compartments . For blocking and dilution buffers, 5% non-fat dry milk in TBST provides optimal results with minimal background . The recommended antibody dilution for Western blotting is 1:1000, combined with appropriate HRP-conjugated secondary antibodies also at 1:1000 dilution . Exposure times between 1-3 minutes are typically sufficient for visualization of the 75 kDa HSPA12A band, though this may vary depending on expression levels in different tissues . When performing co-immunoprecipitation experiments to detect HSPA12A interactions, antibody concentrations should be increased to 1:50 dilution for effective capture of HSPA12A complexes . For examining nucleotide-dependent interactions, inclusion of controlled concentrations of ATP or ADP (0.5-5 mM) in the binding reaction is critical, as these significantly modulate HSPA12A's binding affinity to its partners .

How should samples be prepared for optimal HSPA12A immunodetection?

Sample preparation for optimal HSPA12A immunodetection varies by application but should follow specific guidelines to preserve protein integrity and maintain native interactions. For Western blotting, comprehensive protein extraction requires consideration of HSPA12A's dual localization patterns. Extraction should include both Triton X-100 soluble fractions (containing cytoplasmic HSPA12A) and insoluble fractions (containing nuclear HSPA12A) . For immunohistochemistry applications, paraffin-embedded tissues require heat-mediated antigen retrieval with Tris/EDTA buffer (pH 9.0) to expose HSPA12A epitopes that may be masked during fixation procedures . For immunofluorescence microscopy of cultured cells, fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 preserves the structural integrity while allowing antibody access to intracellular HSPA12A . When examining HSPA12A-SorLA interactions, cell lysis conditions must preserve protein-protein interactions; overnight incubation of lysates with immobilized GST-fusion proteins at 4°C has proven effective for pull-down assays . For nucleotide-dependent studies, samples should be prepared in buffers containing controlled concentrations of ATP or ADP (0.5-5 mM) to examine how these nucleotides regulate HSPA12A binding dynamics with interaction partners .

How does the ADP/ATP binding state affect HSPA12A interactions with SorLA?

The nucleotide-binding state of HSPA12A significantly modulates its interaction with SorLA through conformational changes in the N-terminal ATPase domain. Like other HSP70 proteins, HSPA12A exists in two distinct conformational states dictated by whether ATP or ADP is bound to the N-terminal ATPase domain . GST pull-down assays have definitively demonstrated that increasing ADP concentrations (from 0.5 mM to 5 mM) progressively enhance HSPA12A's binding to SorLA, resulting in greater precipitation of SorLA from cell lysates . Conversely, increasing ATP concentrations (from 0.5 mM to 5 mM) significantly decrease this interaction . Quantitative analysis using one-way ANOVA followed by post-hoc Tukey multiple comparison tests confirmed the statistical significance of these differences (0.5 mM ADP vs 5 mM ADP, p = 0.0178; 0.5 mM ATP vs 5 mM ATP, p = 0.001) . This nucleotide-dependent regulation follows the established paradigm for HSP70 proteins, where ADP-bound conformations exhibit elevated substrate binding activity . Consequently, experimental designs investigating HSPA12A-SorLA interactions must carefully control the nucleotide environment, as the ADP:ATP ratio will significantly impact binding kinetics and could potentially influence downstream trafficking events mediated by this interaction .

Which specific domains of HSPA12A and SorLA mediate their interaction?

The interaction between HSPA12A and SorLA involves specific structural domains and amino acid motifs in both proteins. From the HSPA12A perspective, the 163 C-terminal amino acids of HSPA12A are sufficient to mediate binding to the SorLA cytoplasmic domain (cd), as demonstrated in Y2H screens . Within the SorLA-cd, targeted mutagenesis studies have identified critical acidic amino acid clusters that are essential for HSPA12A binding. Specifically, the pentameric acidic cluster (E34D35D36E37D38) and the C-terminal di-aspartic cluster (D47D48) in the SorLA-cd are crucial for the interaction with HSPA12A . Mutating either of these acidic clusters to alanine residues strongly represses the binding signal in Y2H assays . Interestingly, the C-terminal di-aspartic cluster (D47D48) represents an overlapping binding motif that is also recognized by GGA2, suggesting potential competitive interactions between these adaptor proteins . Deletion experiments further confirmed that the acidic-rich amino acid region from the glycine residue at position 29 to the proline at position 50 in the SorLA-cd is critical for HSPA12A binding . This molecular recognition mechanism appears to be highly specific to HSPA12A, as the closely related HSPA12B protein fails to interact with the SorLA-cd despite structural similarity .

How can false-positive and false-negative results be identified when working with HSPA12A antibodies?

Identifying false-positive and false-negative results when working with HSPA12A antibodies requires systematic controls and validation approaches. For false-positives, secondary antibody-only controls are essential—applying just the HRP-conjugated secondary antibody without primary HSPA12A antibody helps identify non-specific binding . Additionally, using tissue or cell lysates known to lack HSPA12A expression provides important negative controls; untransfected HEK293 cells have successfully served this purpose in pull-down assays . When examining HSPA12A-SorLA interactions, GST-only controls help distinguish specific binding from non-specific protein adherence to the GST tag or beads . For preventing false-negatives, ensuring proper antigen retrieval is critical—heat-mediated antigen retrieval with Tris/EDTA buffer at pH 9.0 has been validated for HSPA12A detection in paraffin-embedded tissues . When studying nucleotide-dependent interactions, controlled concentrations of both ATP and ADP should be tested, as HSPA12A binding affinity varies significantly between these states . When analyzing subcellular localization, examining both Triton X-100 soluble and insoluble fractions is necessary, as HSPA12A distributes between cytoplasmic and nuclear compartments . Validation using multiple detection methods (Western blot, immunoprecipitation, immunohistochemistry) provides convergent evidence of HSPA12A presence and can help identify method-specific detection issues .

What are the common experimental pitfalls when studying HSPA12A-SorLA interactions?

Several common experimental pitfalls can complicate studies of HSPA12A-SorLA interactions. First, neglecting the nucleotide dependence of the interaction represents a major pitfall—experiments lacking controlled ATP/ADP concentrations may yield inconsistent binding results, as HSPA12A's affinity for SorLA varies significantly between ATP and ADP-bound states . Second, overlooking HSPA12A's dual subcellular localization can lead to incomplete extraction and analysis; comprehensive extraction protocols must account for both soluble (cytoplasmic) and insoluble (nuclear) fractions . When performing co-localization studies, temporal dynamics present another challenge—HSPA12A and SorLA co-localize predominantly in perinuclear compartments but show distinct localization in vesicles distally located from the nucleus, suggesting spatial regulation that could be missed in static imaging approaches . Competition between adaptor proteins represents another potential complication—the C-terminal di-aspartic cluster (D47D48) in SorLA-cd serves as an overlapping binding motif for both HSPA12A and GGA2, potentially leading to competitive binding that could confound interaction studies . Finally, specificity controls are essential—the closely related HSPA12B fails to interact with SorLA-cd despite structural similarity to HSPA12A, highlighting the importance of validating antibody specificity when examining these interactions .

How should researchers interpret variations in HSPA12A localization across different cell types?

Interpreting variations in HSPA12A localization across different cell types requires consideration of several biological and methodological factors. First, cell-type specific expression patterns of interaction partners, particularly SorLA, significantly influence HSPA12A localization. In primary cortical astrocytes, HSPA12A demonstrates both nuclear and cytoplasmic distribution with enrichment in perinuclear compartments and vesicular structures . This dual localization pattern is consistent with other HSP70 family members but may vary based on the relative abundance of binding partners like SorLA . When analyzing co-localization with SorLA, researchers should note the spatial heterogeneity—HSPA12A and SorLA primarily co-localize in perinuclear compartments but occupy separate compartments in vesicles distally located from the nucleus . This suggests that the interaction is spatially regulated within cells and may reflect different functional states or trafficking pathways. Experimentally, verifying localization patterns requires complementary approaches; Western blotting of subcellular fractions can confirm the presence of HSPA12A in different cellular compartments, while immunofluorescence microscopy provides spatial resolution . Cell-specific differences in nucleotide metabolism may also influence HSPA12A localization, as its interaction with partners depends on the ATP/ADP binding state . Finally, expression levels achieved in different experimental systems (endogenous expression versus overexpression) can affect localization patterns and should be considered when comparing results across different cell types or experimental conditions .

What emerging techniques might enhance detection and functional analysis of HSPA12A?

Several emerging techniques show promise for enhancing HSPA12A detection and functional analysis. Proximity ligation assays (PLAs) could provide superior sensitivity and spatial resolution for detecting HSPA12A-SorLA interactions in situ, offering quantitative assessment of protein-protein interactions at endogenous expression levels. CRISPR-Cas9 genome editing for endogenous tagging of HSPA12A would enable live-cell imaging with physiological expression levels, avoiding artifacts associated with overexpression systems. For studying the nucleotide-dependent conformational dynamics of HSPA12A, hydrogen-deuterium exchange mass spectrometry (HDX-MS) could reveal structural changes associated with ATP versus ADP binding states that modulate interaction with SorLA. Single-molecule imaging techniques would allow real-time visualization of HSPA12A-SorLA trafficking dynamics in living cells, potentially uncovering how this interaction influences receptor endocytosis and vesicular transport. Cryo-electron microscopy could provide structural insights into the HSPA12A-SorLA complex, particularly focusing on how the critical acidic clusters in SorLA-cd interact with HSPA12A. For high-throughput screening applications, bioluminescence resonance energy transfer (BRET) assays could facilitate screening of compounds that modulate HSPA12A-SorLA interactions, potentially identifying pharmacological tools to manipulate this pathway .

How might HSPA12A research contribute to understanding neurodegenerative diseases?

HSPA12A research holds significant potential for advancing our understanding of neurodegenerative diseases through several mechanistic pathways. First, HSPA12A's selective interaction with SorLA (SORL1) establishes a direct link to Alzheimer's disease pathogenesis, as SORL1 variants are established risk factors for late-onset Alzheimer's disease . By regulating SorLA internalization and subcellular trafficking, HSPA12A may influence amyloid precursor protein (APP) processing and Aβ production, core pathological processes in Alzheimer's disease. The presence of HSPA12A in human cerebral cortex tissue and its detection in fetal brain lysates suggests a neurological function that warrants investigation in neurodegenerative contexts . HSPA12A's expression in astrocytes is particularly noteworthy, as astrocyte dysfunction is increasingly recognized as a contributor to neurodegeneration . The ADP/ATP-dependent interaction mechanism may connect HSPA12A function to cellular energy metabolism, which is frequently dysregulated in neurodegenerative conditions . As a heat shock protein family member, HSPA12A may participate in proteostasis networks that prevent protein aggregation, a hallmark of many neurodegenerative diseases. Furthermore, understanding how HSPA12A affects vesicular trafficking could provide insights into synaptic dysfunction and neuronal connectivity loss in conditions like Alzheimer's disease, Parkinson's disease, and frontotemporal dementia .

What considerations apply when designing experiments to study HSPA12A in different model systems?

Designing experiments to study HSPA12A across different model systems requires careful consideration of several factors to ensure valid and translatable results. First, endogenous expression patterns must be characterized in each model system, as HSPA12A exhibits tissue-specific expression that varies across species; Western blotting has confirmed expression in human fetal kidney, mouse spleen, and rat heart tissues . For cell culture models, primary cortical astrocytes have demonstrated endogenous HSPA12A expression and co-localization with SorLA, making them appropriate for studying native interactions . When establishing overexpression systems, researchers should consider both transient and stable expression approaches; stable single and double transfected HEK293 cell lines overexpressing SorLA, Sortilin, and HSPA12A have been successfully generated for interaction studies . For in vivo models, immunohistochemical validation of HSPA12A expression is essential before proceeding with functional studies; human cerebral cortex tissue has shown positive cytoplasmic staining for HSPA12A . When designing protein-protein interaction studies across different systems, the nucleotide dependence of HSPA12A-SorLA binding must be accounted for, as ADP/ATP ratios may vary across tissues and model organisms . For co-localization studies, the subcellular distribution patterns observed in one cell type (e.g., perinuclear enrichment in astrocytes) should not be automatically assumed in other cell types without verification . Lastly, when translating between in vitro and in vivo models, consideration should be given to how differences in SorLA trafficking dynamics might influence HSPA12A function in different physiological contexts .