HSPA13 Antibody

What is HSPA13 and what cellular functions does it regulate?

HSPA13 (Heat Shock Protein Family A Member 13) is a member of the heat shock protein family that plays critical roles in several cellular processes. Research has identified HSPA13 as essential for plasma cell (PC) differentiation and antibody secretion . At the molecular level, HSPA13 interacts with proteins in the endoplasmic reticulum (ER), particularly Bcap31, to positively regulate protein transport from the ER to the cytosol . Additionally, HSPA13 functions as a checkpoint regulator in the TNFα signaling pathway, where it enhances TNFα-induced recruitment of RIP1 to TNFR1, consequently promoting downstream NF-κB transcriptional responses while attenuating programmed cell death . This dual role positions HSPA13 as a fine-tuner of cellular responses, balancing survival versus death decisions in response to inflammatory stimuli.

How is HSPA13 expression regulated in different cell types and disease states?

HSPA13 exhibits differential expression patterns across cell types, with particularly noteworthy expression in immune cells. Studies have demonstrated that plasmablasts (PBs) and plasma cells (PCs) express high levels of HSPA13, while naïve B cells and germinal center (GC) B cells maintain relatively low expression levels . In disease contexts, HSPA13 mRNA is significantly upregulated in B220+ cells from patients with multiple myeloma (MM) or systemic lupus erythematosus (SLE) . During B cell activation, HSPA13 mRNA increases in LPS-stimulated plasmablasts and plasma cells, suggesting its expression is dynamically regulated during immune cell differentiation and activation . The increased expression in autoimmune conditions points to a potential pathogenic role, making HSPA13 a promising target for therapeutic intervention in diseases characterized by abnormal plasma cell production.

What are the validated applications for HSPA13 antibodies in immunological research?

HSPA13 antibodies have been successfully employed in multiple immunological research applications with specific methodological considerations for each technique:

• Western Blotting: HSPA13 antibodies (such as Proteintech Group Inc. Cat no. 12667-2-AP) have been validated for detecting HSPA13 protein (typically run on 10% SDS-polyacrylamide gels) using a 1:1,000 dilution in TBS-T containing 5% bovine serum albumin . Detection protocols typically utilize HRP-conjugated secondary antibodies and ECL detection systems.

• Immunoprecipitation (IP): HSPA13 antibodies effectively precipitate HSPA13 and its interacting proteins. Protocols involve cell lysis with IP Lysis Buffer, pre-clearing with protein A-sepharose, incubation with undiluted anti-HSPA13 antibody, and collection of protein complexes by centrifugation followed by multiple washing steps .

• Immunohistochemistry (IHC): HSPA13 antibodies can be used to assess protein localization in tissue sections, especially in analyzing disease models such as lupus-prone mice .

• Co-immunoprecipitation: HSPA13 antibodies have been instrumental in identifying protein-protein interactions, particularly with ER proteins like Bcap31, providing insights into HSPA13's functional mechanisms .

What protein extraction methods yield optimal results when detecting HSPA13 by Western blot?

For optimal HSPA13 detection by Western blot, researchers should consider the following extraction protocol:

• Cell Lysis: Use IP Lysis Buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 0.5% NP-40, with appropriate protease and phosphatase inhibitors . For enhanced detection of post-translational modifications, add 5 mM N-ethylmaleimide (NEM).

• Protein Quantification: Standardize protein concentrations (typically 25 μg per lane) to ensure comparable loading .

• Sample Preparation: Mix proteins with SDS loading buffer and heat at 95°C for 5 minutes to ensure complete denaturation.

• Gel Separation: HSPA13 protein is effectively resolved on 10% SDS-polyacrylamide gels .

• Transfer Conditions: Transfer proteins to PVDF membranes using standard protocols with careful optimization of transfer time and current to ensure complete transfer of proteins in the HSPA13 molecular weight range.

• Blocking: Block membranes for 1 hour in 5% fat-free dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) at room temperature .

• Antibody Incubation: For primary incubation, use HSPA13 antibody at 1:1,000 dilution in TBS-T with 5% BSA overnight at 4°C .

This optimized protocol enables sensitive and specific detection of HSPA13 protein in various cell and tissue types.

How do HSPA13 knockout models affect antibody production and autoimmune disease progression?

HSPA13 knockout models have provided compelling evidence for its critical role in antibody production and autoimmune disease development:

These findings collectively demonstrate that HSPA13 is essential for normal plasma cell development and function, while suggesting that targeting HSPA13 could represent a novel therapeutic approach for diseases characterized by aberrant plasma cell activity, including autoimmune conditions and multiple myeloma.

What molecular mechanisms explain HSPA13's role in plasma cell function and antibody secretion?

HSPA13 facilitates plasma cell function and antibody secretion through several interconnected molecular mechanisms:

• Endoplasmic Reticulum (ER) Protein Transport: HSPA13 interacts with ER proteins, particularly Bcap31, to positively regulate protein transport from the ER to the cytosol . This function is crucial for plasma cells, which require extensive ER development and secretory capacity to produce large quantities of antibodies.

• TNFα Signaling Modulation: HSPA13 enhances TNFα-induced recruitment of RIP1 to TNFR1, promoting NF-κB activation . This signaling pathway is important for plasma cell survival and function, as NF-κB regulates genes involved in inflammatory responses and cell survival.

• Apoptosis Regulation: By attenuating the participation of RIP1 in cytosolic complex II, HSPA13 prevents cells from undergoing programmed death (both apoptosis and necroptosis) . This protective function is particularly important for long-lived plasma cells.

• B Cell Differentiation: High expression of HSPA13 in plasmablasts and plasma cells, but not in naïve B cells or germinal center B cells, suggests its involvement in the differentiation process toward antibody-secreting cells .

This multifaceted role positions HSPA13 as a critical regulator of plasma cell biology, affecting both the cellular machinery required for antibody production and the signaling pathways that maintain plasma cell survival and function.

What are the most effective immunoprecipitation protocols for studying HSPA13 protein interactions?

For optimal investigation of HSPA13 protein interactions, researchers should consider the following immunoprecipitation protocol:

• Cell Preparation: Start with approximately 10^7 cells per condition to ensure sufficient protein yield.

• Lysis Conditions: Lyse cells with IP Lysis Buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.5, 0.5% Triton X-100, 0.5% NP-40, 5 mM glycero-2-phosphate, 10 mM NaF, 10% glycerol, and protease/phosphatase inhibitors) . For detection of ubiquitinated proteins or other post-translational modifications, add 5 mM N-ethylmaleimide (NEM).

• Pre-clearing: To reduce non-specific binding, pre-clear 500 μl of lysate with 100 μl protein A-sepharose CL4B by incubating overnight at 4°C with constant shaking .

• Antibody Incubation: After separating the protein A-sepharose by centrifugation, incubate the resulting supernatant with 20 μl undiluted anti-HSPA13 antibody (Proteintech Group Inc. Cat no. 12667-2-AP) and 100 μl fresh protein A-sepharose at room temperature with continuous shaking for 4 hours .

• Washing: Wash the collected pellet 6 times with 100 μl PBS (10 mM Na-phosphate buffer, pH 7.4, 140 mM NaCl) to remove non-specifically bound proteins .

• Elution and Analysis: Elute bound proteins with SDS sample buffer and analyze by SDS-PAGE followed by Western blotting or mass spectrometry to identify interacting partners.

For co-immunoprecipitation studies specifically examining the HSPA13-Bcap31 interaction, researchers can use recombinant plasmids expressing Hspa13-V5 and Bcap31-Flag co-transfected into 293T cells using appropriate transfection reagents .

How can researchers effectively design CRISPR-Cas9 experiments to study HSPA13 function?

For effective CRISPR-Cas9-mediated investigation of HSPA13 function, researchers should follow this comprehensive approach:

• Guide RNA Design:

  • Select target sequences in HSPA13 exons that are critical for protein function

  • Consider using validated guide RNA sequences such as 5′-ATTGTTCTGTTGGGGTG-3′

  • Design multiple gRNAs targeting different regions to increase knockout efficiency

  • Verify target specificity using bioinformatic tools to minimize off-target effects

• Vector Construction:

  • Subclone the guide RNA sequence into a CRISPR-Cas9 vector containing a puromycin resistance selection marker

  • For conditional knockout studies in specific cell types, consider using cell-type specific promoters or Cre-loxP systems (as demonstrated in the CD19-cre mediated B cell-specific deletion model)

• Cell Transfection and Selection:

  • Transfect target cells (such as HT29 or Jurkat cells) with the CRISPR-Cas9 construct

  • Select transfected cells with puromycin (1 μg/ml) for 48-72 hours

  • Isolate single colonies for expansion and validation

• Knockout Verification:

  • Confirm gene disruption by DNA sequencing of the targeted region

  • Verify protein elimination by Western blotting using validated HSPA13 antibodies

  • Assess mRNA levels by RT-PCR using appropriate primers

• Functional Validation:

  • For plasma cell studies, assess antibody production using ELISA

  • For TNFα signaling studies, examine NF-κB activation and cell death responses

  • Compare knockout cells with wild-type controls under various stimulation conditions

• In vivo Applications:

  • For tissue-specific studies, consider conditional knockout approaches as demonstrated with the B cell-specific HSPA13 deletion (CD19-cre mediated)

  • Validate phenotypes in disease models such as pristane-induced lupus or MRL/lpr models

This comprehensive approach enables robust investigation of HSPA13 function in various cellular contexts and experimental systems.

How can single-cell RNA-sequencing approaches be optimized to study HSPA13 expression in heterogeneous immune cell populations?

Optimizing single-cell RNA-sequencing for HSPA13 expression analysis in immune cell populations requires attention to several methodological considerations:

• Sample Preparation:

  • Isolate cells in PBS containing 0.04% bovine serum albumin to maintain cell viability and minimize stress responses that might alter HSPA13 expression

  • Aim for final concentration of approximately 200 cells per μL for optimal capture efficiency

  • Process samples immediately to prevent ex vivo changes in gene expression profiles

• Platform Selection:

  • Use high-throughput platforms like 10X Genomics Chromium for capturing large numbers of cells to ensure representation of rare cell populations

  • Consider paired analysis with VDJ-sequencing to correlate HSPA13 expression with B cell receptor repertoire in studies examining antibody production

• Sequencing Depth:

  • Aim for minimum coverage of 50,000-100,000 reads per cell to ensure detection of HSPA13 transcripts, which may be expressed at moderate levels

  • Balance between cell number and sequencing depth based on the specific research question

• Data Analysis Optimization:

  • Use appropriate normalization methods that account for technical variables

  • Implement batch correction when combining multiple samples

  • Apply dimensionality reduction techniques (t-SNE, UMAP) to visualize cell populations

  • Include feature plots specifically highlighting HSPA13 expression across identified clusters

• Validation Strategies:

  • Confirm key findings with orthogonal methods such as flow cytometry or immunohistochemistry

  • Perform RNA velocity analysis to identify trajectories of cellular differentiation and HSPA13 expression dynamics

  • Validate cell type identification using established marker genes alongside HSPA13

• Integration with Protein Data:

  • Consider complementary CITE-seq approaches to simultaneously measure cell surface proteins and HSPA13 mRNA

  • Correlate HSPA13 mRNA levels with functional antibody secretion metrics

This optimized approach allows researchers to comprehensively characterize HSPA13 expression patterns across diverse immune cell subsets and developmental stages, providing insight into its regulation and function in normal and pathological conditions.

What are the challenges and solutions in developing specific inhibitors targeting HSPA13 for potential therapeutic applications?

Developing specific HSPA13 inhibitors presents several challenges and potential solutions:

• Target Specificity Challenges:

  • HSPA13 belongs to the heat shock protein family, which shares significant sequence homology among members

  • Solution: Focus on unique structural features or binding pockets specific to HSPA13 through detailed structural biology studies

  • Approach: Employ structure-guided design using crystal structures or homology models of HSPA13, particularly focusing on regions that interact with specific partners like Bcap31

• Functional Assay Development:

  • Challenge: Creating high-throughput screening assays that specifically measure HSPA13 activity

  • Solution: Develop assays based on HSPA13's role in protein transport from ER to cytosol

  • Methods: Design fluorescent reporter systems monitoring protein transport, or FRET-based assays measuring HSPA13-Bcap31 interactions

• Delivery to Target Cells:

  • Challenge: Ensuring inhibitors reach B cells and plasma cells in sufficient concentrations

  • Solution: Consider B cell-targeting delivery systems or antibody-drug conjugates

  • Approach: Leverage insights from CD19-targeted therapies already in clinical use

• Balancing Efficacy and Safety:

  • Challenge: HSPA13 plays roles in both pathological and normal cellular processes

  • Solution: Tune inhibitor potency to reduce pathological plasma cell activity without completely eliminating normal antibody responses

  • Strategy: Test HSPA13 inhibitors in models like the pristane-induced lupus or MRL/lpr mice to establish therapeutic windows

• Evaluation in Disease Models:

  • Challenge: Selecting appropriate models that recapitulate human disease features

  • Solution: Use combination of in vitro systems with patient-derived cells and in vivo models

  • Approach: Test candidates in both pristane-induced lupus and genetic MRL/lpr models as demonstrated in HSPA13 knockout studies

• Biomarker Development:

  • Challenge: Monitoring treatment efficacy in clinical settings

  • Solution: Develop assays measuring HSPA13 activity or downstream effects

  • Methods: Quantify antibody-secreting cell numbers, serum autoantibody levels, and NF-κB activation status in response to treatment

These strategies provide a framework for overcoming the challenges inherent in developing HSPA13-targeted therapeutics for conditions characterized by abnormal plasma cell activity.

What are common pitfalls in HSPA13 antibody-based experiments and how can they be avoided?

Researchers working with HSPA13 antibodies may encounter several technical challenges. Here are common pitfalls and their solutions:

• Non-specific Binding in Western Blots:

  • Pitfall: Multiple bands or high background obscuring HSPA13-specific signal

  • Solution: Optimize blocking conditions using 5% fat-free dry milk in TBS-T for 1 hour at room temperature

  • Additional step: Increase washing frequency to 4-5 times with TBS-T for 5 minutes each

  • Validation: Always include positive controls (cells known to express HSPA13, like plasma cells) and negative controls (HSPA13 knockout cells)

• Inefficient Immunoprecipitation:

  • Pitfall: Poor yield of HSPA13 protein in IP experiments

  • Solution: Pre-clear lysates with protein A-sepharose to reduce non-specific binding

  • Optimization: Increase antibody amount (up to 20 μl undiluted) and extend incubation time to 4 hours

  • Technique: Ensure thorough washing (6 times with PBS) while maintaining gentle conditions to preserve protein-protein interactions

• Variable HSPA13 Detection in Different Cell Types:

  • Pitfall: Inconsistent HSPA13 signal across cell types

  • Solution: Recognize that HSPA13 expression varies significantly, with high levels in plasma cells but low levels in naïve B cells

  • Approach: Adjust exposure times based on expected expression levels in different cell populations

  • Control: Include β-tubulin or other housekeeping proteins as loading controls

• Challenges in Co-Immunoprecipitation Studies:

  • Pitfall: Failure to detect interacting partners

  • Solution: Use mild lysis conditions (0.5% Triton X-100, 0.5% NP-40) to preserve protein-protein interactions

  • Alternative: Consider crosslinking approaches for transient interactions

  • Validation: Use recombinant tagged proteins (Hspa13-V5, Bcap31-Flag) in overexpression systems to validate interactions before attempting endogenous detection

• Antibody Batch Variation:

  • Pitfall: Different results with different antibody lots

  • Solution: Validate each new antibody lot against previous standards

  • Approach: Maintain positive control lysates from characterized experiments for comparison

  • Documentation: Record antibody lot numbers and specific dilutions in laboratory records

By anticipating these challenges and implementing appropriate controls and optimizations, researchers can significantly improve the reliability and reproducibility of HSPA13 antibody-based experiments.

How can researchers reconcile conflicting data about HSPA13 function in different experimental systems?

When facing conflicting data about HSPA13 function across experimental systems, researchers should employ the following systematic approach:

• Context-Dependent Function Analysis:

  • Recognize that HSPA13 functions differently across cell types, with distinct roles in B cells versus other cell types

  • Systematically document and compare experimental conditions, including cell types, activation states, and stimulation protocols

  • Establish a matrix comparing HSPA13 functions across different cellular contexts to identify patterns

• Methodology Reconciliation:

  • Compare knockout strategies: conditional knockouts (like CD19-cre B cell-specific deletion) versus complete knockout or CRISPR-generated knockouts

  • Evaluate differences between in vitro cellular systems and in vivo mouse models

  • Consider acute versus chronic loss of HSPA13 function, as compensatory mechanisms may develop in stable knockout lines

• Dose-Dependent Effects Assessment:

  • Investigate whether HSPA13 exhibits threshold effects by using heterozygous models or partial knockdown approaches

  • Employ inducible systems to control the timing and degree of HSPA13 expression

  • Compare overexpression phenotypes with knockout phenotypes to establish a complete functional spectrum

• Pathway Integration Analysis:

  • Recognize that HSPA13 participates in multiple pathways: B cell differentiation, TNFα signaling, and ER protein transport

  • Employ pathway-specific readouts to determine which functions are preserved or altered in different experimental contexts

  • Use chemical inhibitors of specific pathways to isolate the contribution of HSPA13 to each process

• Technical Validation Strategy:

  • Implement multiple independent techniques to verify key findings

  • For antibody-related findings, use multiple antibody clones targeting different epitopes

  • Complement genetic approaches (knockouts) with biochemical approaches (inhibitors or dominant negative constructs)

• Multi-Model Verification:

  • Test critical findings across multiple model systems: cell lines, primary cells, and in vivo models

  • Consider species differences when comparing mouse and human systems

  • Validate findings in disease-relevant models such as lupus-prone mice

By systematically addressing these aspects, researchers can develop an integrated understanding of HSPA13 function that accommodates seemingly conflicting data from different experimental contexts.