HSPA5 Monoclonal Antibody

What is HSPA5 and why is it an important research target?

HSPA5 (Heat Shock Protein Family A Member 5), also known as GRP78 or BiP, is a 72-78 kDa protein primarily localized in the endoplasmic reticulum lumen. It functions as a molecular chaperone involved in protein folding and assembly, and plays critical roles in the cellular stress response, particularly endoplasmic reticulum (ER) stress. Its expression is maintained or increased during cellular stress conditions, making it a valuable biomarker for various pathological states including cancer, neurodegenerative diseases, and inflammatory conditions. The protein is widely expressed across multiple tissues including brain, liver, muscle, and various carcinoma types, creating numerous research applications .

What are the typical applications for HSPA5 monoclonal antibodies in research?

HSPA5 monoclonal antibodies have been validated for multiple experimental applications including:

• Western Blot (WB) analysis of cell and tissue lysates

• Immunohistochemistry (IHC) on paraffin-embedded tissues

• Immunocytochemistry (ICC) for cellular localization studies

• Immunofluorescence (IF) for high-resolution visualization
  These applications enable researchers to detect, quantify, and localize HSPA5 in various experimental systems, facilitating studies on stress responses, cancer biology, and protein quality control mechanisms .

Which tissue samples have been verified for HSPA5 antibody reactivity?

Based on published validation data, HSPA5 monoclonal antibodies have been successfully tested on:

• Human cell lines: HeLa (cervical carcinoma), Jurkat (T-cell leukemia), MCF-7 (breast cancer)

• Tissue samples: Human colon carcinoma, human breast carcinoma, rat liver

• Other experimentally confirmed tissues expressing HSPA5 include articular cartilage, brain, fibroblasts, muscle, melanoma, and liver
  This broad reactivity profile enables comparative studies across different tissue types and pathological conditions .

What are the optimal dilutions and conditions for Western blot detection of HSPA5?

For Western blot applications, the recommended working dilutions typically range from 1:1000 to 1:2000, though optimal concentrations should be determined empirically for each experimental system. For optimal results:

• Use PVDF membrane for protein transfer

• Apply 1-10 μg/mL of HSPA5 antibody depending on the specific clone

• Perform detection under reducing conditions

• Include appropriate positive controls (such as HeLa, Jurkat, or MCF-7 cell lysates)

• Expect to observe a specific band at approximately 72-78 kDa

The molecular weight discrepancy (72 vs. 78 kDa) observed in different detection systems may result from post-translational modifications or differences in electrophoresis conditions .

How should HSPA5 antibodies be stored and handled to maintain optimal activity?

To preserve antibody integrity and activity:

• Store concentrated stock at -20°C for long-term storage (up to one year)

• For frequent use, aliquot and store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles which can cause protein denaturation and loss of binding activity

• Most HSPA5 antibodies are supplied in a buffer containing phosphate-buffered saline (pH 7.4), 150mM NaCl, 0.02% sodium azide, and 50% glycerol with stabilizing proteins (0.4-0.5mg/ml BSA)

• Allow antibodies to equilibrate to room temperature before opening the vial to prevent condensation .

What are effective approaches for validating HSPA5 antibody specificity in new experimental systems?

A comprehensive validation strategy should include:

• Positive and negative control tissues with known HSPA5 expression patterns

• Parallel detection with alternative antibody clones targeting different epitopes

• Signal correlation with HSPA5 mRNA expression data

• Blocking peptide competition assays when available

• siRNA knockdown or CRISPR knockout of HSPA5 as gold-standard negative controls

• Western blot analysis to confirm a single band of appropriate molecular weight

These validation steps are particularly important when extending the use of the antibody to previously untested species or tissue types .

How can non-specific binding be minimized in HSPA5 immunodetection experiments?

Non-specific binding is a common challenge with antibody-based detection. To minimize this issue:

• Optimize blocking conditions (typically 5% non-fat dry milk or BSA)

• Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

• Titrate primary antibody concentration to determine optimal signal-to-noise ratio

• Include 0.1-0.3% Triton X-100 in blocking buffers for membranous proteins

• Consider using monoclonal rather than polyclonal antibodies for higher specificity

• Ensure all washing steps are thorough (at least 3-5 washes of 5-10 minutes each)

For Simple Western™ systems specifically, be aware that some HSPA5 antibodies may show non-specific interaction with the 230 kDa standard, requiring additional optimization steps .

What are the key considerations for cross-species reactivity when using HSPA5 antibodies?

While HSPA5 is highly conserved across species, antibody cross-reactivity should not be assumed without validation:

• Confirm the specific species reactivity listed for each antibody clone (common reactivities include human, mouse, and rat)

• When testing in unlisted species (e.g., non-human primates), perform preliminary validation experiments

• Compare amino acid sequence homology in the epitope region between target species

• Consider using epitope-mapped antibodies for greater predictability of cross-species reactivity

• Modify experimental conditions (antibody concentration, incubation time, temperature) when adapting to new species

As noted in customer inquiries, some HSPA5 antibodies that work in human samples have been successfully applied to monkey tissues, though formal validation is recommended .

How can researchers address variability in HSPA5 detection across different tissue types?

Tissue-specific factors can significantly influence HSPA5 detection. To address this variability:

• Optimize fixation conditions for each tissue type (fixation time, fixative composition)

• Adjust antigen retrieval methods (heat-induced vs. enzymatic retrieval)

• Modify antibody concentration based on target tissue expression levels

• Consider tissue-specific blocking agents to reduce background

• Validate with tissue-specific positive and negative controls

• Normalize expression data to appropriate housekeeping proteins for quantitative analyses

HSPA5 expression varies significantly across tissues, with particularly high levels observed in secretory cells and tissues under ER stress conditions .

How can HSPA5 antibodies be utilized to investigate endoplasmic reticulum stress responses?

HSPA5 serves as a key marker and regulator of ER stress responses. Advanced applications include:

• Temporal analysis of HSPA5 induction following ER stress-inducing treatments

• Co-immunoprecipitation studies to identify stress-dependent protein interactions

• Chromatin immunoprecipitation (ChIP) to study transcription factor binding to the HSPA5 promoter

• Proximity ligation assays to visualize protein-protein interactions in situ

• Subcellular fractionation combined with immunoblotting to track HSPA5 translocation during stress

• Multiplexed immunofluorescence to correlate HSPA5 expression with other UPR pathway components

These approaches can provide mechanistic insights into cellular adaptation to proteotoxic stress and identify potential therapeutic targets .

What is the significance of the Hspa5 promoter in recombinant protein production, and how can it be studied?

The Hspa5 promoter has emerged as a valuable tool for stable recombinant protein expression, particularly for monoclonal antibodies. Key research approaches include:

• Promoter-reporter assays to quantify transcriptional activity under various conditions

• Comparison with conventional promoters (CMV, hEF1α) in fed-batch cell culture systems

• Analysis of promoter activity correlation with endoplasmic reticulum stress markers

• Investigation of how the Hspa5 promoter maintains or increases expression in late culture phases

• Assessment of promoter effectiveness across different IgG subclasses and protein targets

• Chromatin structure and epigenetic modification analysis of the integrated promoter

The Hspa5 promoter uniquely enables improved monoclonal antibody productivity per cell in the late culture period, offering advantages for biopharmaceutical manufacturing .

How can researchers integrate HSPA5 detection with analyses of other stress response pathways?

Advanced multiparametric approaches include:

• Multiplexed immunofluorescence or mass cytometry to simultaneously detect HSPA5 with other stress markers

• Correlation of HSPA5 protein levels with transcriptomic profiles of UPR genes

• Phospho-specific detection of stress-activated signaling kinases (PERK, IRE1α) alongside HSPA5

• Single-cell analysis techniques to capture heterogeneity in stress responses

• Integration of HSPA5 detection with apoptosis markers to study stress-induced cell death thresholds

• Temporal studies correlating HSPA5 induction with metabolic adaptations to stress

These integrated approaches can reveal the complex interplay between various cellular stress response mechanisms and their contribution to disease pathogenesis .

What role does HSPA5 play in cancer biology, and how can HSPA5 antibodies facilitate cancer research?

HSPA5 has multifaceted roles in cancer progression and therapeutic resistance. Cutting-edge research applications include:

• Analysis of HSPA5 surface translocation in various cancer types using non-permeabilized immunostaining

• Correlation of HSPA5 expression patterns with patient outcomes and treatment response

• Investigation of HSPA5 as a target for antibody-drug conjugates or CAR-T cell therapy

• Evaluation of HSPA5 as a biomarker for chemotherapy resistance

• Studies of HSPA5 involvement in tumor microenvironment regulation and immune evasion

HSPA5 has been detected in various cancer cell lines including HeLa, Jurkat, and MCF-7, making these useful model systems for such investigations .

How can researchers leverage HSPA5 antibodies for studying neurodegenerative diseases?

Emerging evidence links HSPA5 dysfunction to neurodegenerative conditions. Advanced research strategies include:

• Brain region-specific analysis of HSPA5 expression in disease models and human samples

• Co-localization studies with protein aggregates (Aβ, tau, α-synuclein)

• Investigation of HSPA5 in blood-brain barrier integrity and glial activation

• Assessment of HSPA5 modulation as a neuroprotective strategy

• Analysis of HSPA5 genetic variants and their impact on disease susceptibility

• Temporal studies of HSPA5 expression during disease progression

Positive staining of HSPA5 has been observed in human brain tissue, making it a valuable target for neurodegenerative disease research .

What are the considerations for using HSPA5 antibodies in live-cell imaging and dynamic studies?

For researchers pursuing real-time visualization of HSPA5 dynamics:

• Select antibody formats compatible with live-cell applications (Fab fragments, nanobodies)

• Consider site-specific conjugation strategies to maintain antibody functionality

• Optimize antibody concentration to minimize perturbation of natural HSPA5 function

• Validate antibody specificity in live-cell conditions with appropriate controls

• Combine with fluorescent protein fusions for comparative validation

• Employ super-resolution microscopy techniques for detailed subcellular localization

These approaches enable visualization of dynamic HSPA5 responses to cellular stresses with high temporal and spatial resolution, providing insights into stress response kinetics that are not accessible through fixed-cell methods .