HSPA13 Antibody

What is HSPA13 and why is it significant in immunological research?

HSPA13 is a member of the heat shock protein 70 (Hsp70) family that plays a critical role in plasma cell differentiation and antibody secretion. It has been implicated in several disease processes, including systemic lupus erythematosus (SLE), multiple myeloma (MM), and hepatocellular carcinoma (HCC) . Research significance derives from its:

• Essential function in endoplasmic reticulum (ER) protein processing

• Critical role in plasma cell development and antibody production

• Upregulation in autoimmune disorders and certain cancers

• Potential as a therapeutic target for diseases with pathological plasma cells

Studies with B cell-specific Hspa13 knockout (Hspa13 cKO) mice have demonstrated significant reductions in plasmablasts, plasma cells, and antibody production, confirming its importance in immunological processes .

What is the typical molecular weight detected by HSPA13 antibodies in various applications?

HSPA13 typically appears at 60-70 kDa in Western blot applications despite its calculated molecular weight of 52 kDa (471 amino acids) . This discrepancy may result from:

• Post-translational modifications

• Protein interaction with other molecules

• Methodological variations in sample preparation

Researchers should anticipate seeing bands at approximately 60-70 kDa when using validated HSPA13 antibodies in Western blot applications .

Which cell types and tissues consistently express HSPA13?

HSPA13 expression follows a specific pattern in immune cells:

• High expression in plasmablasts (PBs) and plasma cells (PCs)

• Low expression in naïve B cells and germinal center (GC) B cells

• Increased expression in B220+ cells from patients with multiple myeloma or SLE

In cancer research, HSPA13 has been found highly expressed in:

• Hepatocellular carcinoma tissues (68.7% of cases in one study)

• Human endometrial cancer tissue

• Various HCC cell lines including Huh-7 and SK-Hep-1

How should I validate HSPA13 antibody specificity in my experiments?

Comprehensive validation should include multiple approaches:

• Positive controls: Use HEK-293, HeLa, or K-562 cells, which have been confirmed to express HSPA13

• Knockdown validation: Compare antibody signal between wild-type and HSPA13 knockdown/knockout samples

• Multiple detection methods: Validate findings across techniques (WB, IHC, IF)

• Cross-reactivity testing: Test antibody specificity against other heat shock proteins

• Multiple antibody validation: Use antibodies targeting different epitopes of HSPA13

For gene silencing validation, researchers have successfully used HSPA13-targeting siRNAs in cell lines such as Huh-7 and SK-Hep-1 to confirm antibody specificity .

What are the recommended protocols for HSPA13 immunohistochemistry?

Based on published protocols, optimal IHC conditions include:

• Antigen retrieval: Primary recommendation is TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative

• Antibody dilution: 1:20-1:200 range for most commercial HSPA13 antibodies

• Detection system: Streptavidin-biotin or polymer-based detection systems work well

• Positive control tissues: Human endometrial cancer tissue or HCC samples

• Negative controls: Include sections with isotype control antibodies and HSPA13-low tissues

For immunofluorescence applications, researchers should consider dual staining with ER markers (such as calnexin or PDI) to confirm HSPA13's subcellular localization .

What are the optimal sample preparation methods for detecting HSPA13 in Western blots?

To obtain reliable results when detecting HSPA13 in Western blots:

• Lysis buffer composition: Use RIPA buffer supplemented with protease inhibitors

• Sample handling: Maintain samples at 4°C during preparation to prevent protein degradation

• Protein loading: 20-50 μg of total protein per lane is typically sufficient

• Antibody dilution: Use 1:500-1:3000 dilution range for primary antibody

• Blocking conditions: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Signal development: Both chemiluminescence and fluorescence-based detection systems are suitable

When comparing HSPA13 expression between different samples, consider using β-actin or GAPDH as loading controls.

How can HSPA13 antibodies be used to study protein-protein interactions in the ER?

HSPA13 interacts with several proteins in the ER, making co-immunoprecipitation (co-IP) an important research application:

• Recommended protocol:

  • Crosslink HSPA13 antibody (0.5-4.0 μg) to magnetic beads

  • Incubate with 1.0-3.0 mg of total protein lysate

  • Wash extensively to remove non-specific binding

  • Elute and analyze by Western blot

• Known interactors to investigate:

  • Bcap31 (confirmed interaction partner in the ER)

  • Sec61 translocon complex components

  • TANK (in HCC studies)

• Control recommendations:

  • IgG control to assess non-specific binding

  • Reciprocal co-IP using antibodies against putative binding partners

  • Input controls (10% of lysate used for IP)

Research has demonstrated that HSPA13 interacts with ER proteins to regulate protein transport from the ER to the cytosol, making this methodology particularly valuable for secretory pathway studies .

How should researchers interpret HSPA13 expression differences in disease models?

When studying HSPA13 in disease contexts:

• Autoimmune disease models:

  • Elevated HSPA13 mRNA in B220+ cells from SLE patients correlates with disease activity

  • In Hspa13 cKO mice, reduced autoantibodies and proteinuria were observed in both pristane-induced lupus and MRL/lpr mouse models

  • Data interpretation should consider both cellular HSPA13 levels and downstream antibody production

• Cancer research implications:

  • High HSPA13 expression in HCC predicts poor clinical prognosis and correlates with vascular invasion

  • Functional studies show HSPA13 promotes HCC proliferation, migration, and invasion

  • Mechanistic investigations reveal HSPA13 interacts with TANK to inhibit its ubiquitination and degradation

• Data reconciliation approaches:

  • Compare protein vs. mRNA expression patterns

  • Correlate HSPA13 levels with functional outcomes

  • Consider tissue-specific regulation mechanisms

What are the considerations for studying HSPA13 in protein translocation experiments?

HSPA13's role in protein translocation requires specific experimental approaches:

• Experimental design recommendations:

  • Use reporter proteins like transthyretin (TTR) to monitor translocation efficiency

  • Compare wild-type vs. ATPase-inactive HSPA13 mutants

  • Employ subcellular fractionation to distinguish cytosolic vs. ER protein pools

• Key observations from literature:

  • HSPA13 overexpression inhibits translocation of secreted proteins like TTR

  • ATPase-inactive HSPA13 mutants further inhibit translocation and maturation of secretory proteins

  • Mistargeted secretory proteins accumulate in cytosol when HSPA13 is overexpressed

• Analytical approaches:

  • Use proximity labeling methods (APEX2) to determine protein localization

  • Monitor signal peptide cleavage as indicator of successful translocation

  • Assess proteasomal degradation of mistargeted proteins using inhibitors like MG132

What are common challenges when working with HSPA13 antibodies and how can they be resolved?

Researchers frequently encounter these challenges:

How should researchers interpret contradictory HSPA13 expression data between different techniques?

When facing discrepancies between different detection methods:

• Potential explanations:

  • Epitope accessibility differences between techniques

  • Post-translational modifications affecting antibody binding

  • Different sensitivity thresholds for each methodology

  • Sample preparation variations impacting protein detection

• Recommended reconciliation approaches:

  • Use multiple antibodies targeting different HSPA13 epitopes

  • Employ complementary detection methods (mRNA quantification, protein detection)

  • Include appropriate positive and negative controls for each technique

  • Consider subcellular localization differences detected by various methods

• Validation strategies:

  • Genetic approaches (knockdown/knockout) to confirm specificity

  • Recombinant protein expression as positive controls

  • Comparative analysis across multiple cell lines or tissues

What considerations are important when designing experiments to study HSPA13's role in plasma cell differentiation?

For studying HSPA13 in plasma cell biology:

• Experimental models:

  • LPS-stimulated B cells (in vitro model for plasma cell differentiation)

  • CD19-cre mediated B cell-specific Hspa13 deletion mouse model

  • Atacicept (TACI-IgG) treatment in lupus-prone mice

• Key readouts to measure:

  • Plasmablast (TACI+CD138+B220intCD19int) and plasma cell (TACI+CD138+B220-CD19-) populations by flow cytometry

  • Antibody production following stimulation (LPS in vitro, SRC or NP-immunization in vivo)

  • Class-switched and somatically hypermutated antibodies

  • Affinity maturation assessment

• Controls and variables to consider:

  • Timing of HSPA13 expression during B cell differentiation

  • Correlation between HSPA13 levels and antibody secretion capacity

  • Impact of HSPA13 modulation on ER stress and UPR activation

  • Comparison with other heat shock protein family members

What emerging applications of HSPA13 antibodies show promise for translational research?

Several innovative research directions are emerging:

• Therapeutic target validation:

  • HSPA13 inhibition as a strategy to reduce pathologic plasma cells in autoimmune diseases

  • Potential applications in multiple myeloma treatment where plasma cell reduction is desired

  • Exploration as a target in hepatocellular carcinoma therapy

• Biomarker development:

  • HSPA13 expression as a prognostic indicator in HCC patients

  • Correlation with treatment response in autoimmune conditions

  • Monitoring B cell to plasma cell differentiation in disease contexts

• Mechanistic investigations:

  • HSPA13's role in regulating protein quality control in the ER

  • Interactions with other chaperones and protein transport machinery

  • Structure-function studies to identify critical domains for therapeutic targeting

How might comparative analyses of HSPA13 across species inform antibody selection and experimental design?

Cross-species conservation analysis reveals:

• Evolutionary conservation patterns:

  • HSPA13 shows high conservation across mammalian species

  • The ATPase domain is particularly well-conserved

  • Species-specific variations may impact antibody cross-reactivity

• Antibody selection implications:

  • Antibodies targeting conserved regions may work across multiple species

  • When studying specific species, validate antibody reactivity explicitly for that organism

  • Consider targeting species-specific epitopes for selective detection

• Model system selection guidance:

  • Mouse models show comparable HSPA13 function to human systems

  • Cell line selection should consider endogenous HSPA13 expression levels

  • Potential differences in HSPA13 regulation across species should inform data interpretation

Understanding these cross-species similarities and differences is critical for translating findings between model organisms and human disease applications.