HSPA1A Antibody
Description
Definition and Function of HSPA1A Antibody
The HSPA1A antibody targets the inducible 70 kDa heat shock protein (HSPA1A), which is crucial for maintaining cellular homeostasis during stress conditions such as heat, hypoxia, or infection . This protein stabilizes misfolded proteins, facilitates refolding, and participates in immune modulation by interacting with Toll-like receptors (TLRs) . The antibody is widely used to:
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Detect HSPA1A expression in human, mouse, and rat tissues or cell lines .
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Study its role in cancer progression, immune responses, and stress adaptation .
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Validate protein levels in experimental models of diseases like transient cerebral ischemia or thymoma .
2.1. Western Blot and Immunohistochemistry
The HSPA1A antibody reliably detects a single band at ~66–72 kDa in Western blot assays across species, including:
| Cell Line/Tissue | Species | Molecular Weight (kDa) | Conditions |
|---|---|---|---|
| Jurkat (T-cell leukemia) | Human | 72 | Reducing, heat-shocked |
| C2C12 (myoblast) | Mouse | 66 | Reducing |
| Liver tissue | Human | 67 | Simple Western™ analysis |
2.2. Immune Modulation Studies
HSPA1A antibodies have been instrumental in elucidating the protein’s interaction with immune receptors:
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TLR2/4 Blocking: Pre-treatment with TLR2/4 inhibitors reduces HSPA1A-induced cytokine secretion (e.g., 43% decrease in IL-1β, 65% decrease in TNF-α) .
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Cancer Research: In ovarian cancer, HSPA1A on tumor cells activates neutrophil ROS production via TLR2/4, promoting tumor progression .
3.1. Role in Cancer
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HSPA1A is overexpressed in tumors (e.g., breast, lung, prostate) and correlates with poor patient survival .
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It protects cancer cells from apoptosis and oxidative stress while enhancing immune evasion .
3.2. Immune System Interactions
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Cytokine Regulation: HSPA1A binding to TLR2/4 on monocytes triggers IL-1β, TNF-α, and IL-10 secretion, which is critical in inflammatory responses .
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Viral Entry: Acts as a post-attachment receptor for rotavirus A, facilitating viral entry into cells .
3.3. Therapeutic Implications
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Inhibiting HSPA1A-TLR interactions reduces pro-tumor immune activity, suggesting pathways for targeted therapies .
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Antibodies against HSPA1A are being explored to disrupt its protective role in cancer cells .
Future Directions
Ongoing research focuses on:
Product Specs
Q&A
What experimental approaches validate HSPA1A antibody specificity in Western blotting?
Three orthogonal validation methods are essential:
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Induced expression controls: Compare lysates from heat-shocked (42°C for 30 min + 3h recovery) vs. untreated Jurkat cells, expecting ≥5-fold intensity increase at 70 kDa . Source 1 shows this approach detects a 70 kDa band only in heat-shocked conditions.
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Knockout validation: Use CRISPR-edited cell lines lacking HSPA1A; absence of band confirms target specificity.
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Peptide blocking: Pre-incubate antibody with recombinant HSPA1A (Met1-Asp641 fragment). Complete signal loss indicates epitope specificity .
Table 1: Recommended Western Blot Conditions from Literature
| Parameter | Source 1 | Source 3 | Source 6 |
|---|---|---|---|
| Antibody Conc. | 2 µg/mL | 0.1 µg/mL | 0.1 µg/mL |
| Secondary Antibody | HAF018 (1:5k) | HAF007 (1:10k) | HAF008 (1:10k) |
| Detection System | Chemiluminescence | Chemiluminescence | Chemiluminescence |
| Reducing Conditions | Yes | Yes | Yes |
How does species cross-reactivity impact experimental design for HSPA1A studies?
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Verify transcript variants: Mouse HSPA1A lacks exon 2 in some isoforms, potentially creating false negatives .
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Test tissue-specific glycosylation: Liver samples show 5 kDa larger bands vs. cell lines due to post-translational modifications .
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Confirm absence in non-target species: Negative controls should include at least two phylogenetically distant species (e.g., zebrafish, Drosophila).
What factors cause variability in HSPA1A immunohistochemical staining across tissue types?
Four key variables require optimization:
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Epitope retrieval: Antigen Retrieval Reagent-Basic (pH 9.0, 95°C, 20 min) outperforms proteinase K in kidney cancer sections .
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Fixation duration: Prolonged formalin fixation (>48h) masks epitopes; citrate buffer incubation should extend to 40 min for archival samples.
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Nuclear vs. cytoplasmic localization: Heat shock induces nuclear translocation detectable via counterstain correlation (DAPI for nuclei, NL007 secondary for HSPA1A) .
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Endogenous blocker selection: 5% normal goat serum reduces background in liver tissue better than BSA-based blockers .
How can researchers resolve contradictory data on HSPA1A’s pro-inflammatory vs. immunosuppressive roles?
Source 4 reveals dual functionality through TLR interaction kinetics:
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Early phase (0-6h post-stimulation): HSPA1A binds TLR2/4, activating NF-κB via MyD88, increasing IL-1β (268.6 pg/mL → 115.0 pg/mL with TLR2 blockade) .
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Late phase (12-24h): CD91-mediated endocytosis promotes IL-10 secretion (84.3 pg/mL → 22.1 pg/mL with CD91 inhibition) .
Methodological recommendations:
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Time-course experiments with 3h intervals
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Combinatorial receptor blockade (TLR2 + CD91)
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Single-cell cytokine profiling via RNA-FISH
What advanced techniques characterize HSPA1A’s membrane association dynamics?
Source 5 introduces a lipid-centric workflow:
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Quantitative lipidomics: Heat shock (42°C, 1h) increases phosphatidylserine (PS) by 3.2-fold in plasma membranes .
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RNAi validation: PSS1/PSS2 knockdown reduces HSPA1A membrane localization by 78% (CTCF ratio 0.15 vs. 0.67 in controls) .
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Live-cell imaging: Lact-C2-GFP biosensor confirms PS-HSPA1A co-localization (Pearson’s r = 0.82 post-heat shock) .
Table 2: Lipidomic Changes Impacting HSPA1A Localization
| Lipid Class | Control (mol%) | Heat Shock (mol%) | Δ% | p-value |
|---|---|---|---|---|
| Phosphatidylserine | 8.2 ± 1.1 | 26.3 ± 3.4 | +220.7 | <0.001 |
| Sphingomyelin | 12.4 ± 2.3 | 6.8 ± 1.9 | -45.2 | 0.003 |
| Cholesterol | 31.5 ± 4.1 | 28.7 ± 3.8 | -8.9 | 0.12 |
How should researchers optimize HSPA1A detection in single-cell RNA-seq experiments?
Three critical considerations emerge from recent studies:
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Stress induction timing: Maximum HSPA1A mRNA occurs at 3h post-heat shock (42°C), but protein peaks at 6h . Synchronize scRNA-seq with proteomic sampling.
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Batch effect control: Spike-in ERCC RNA standards (1:1000 dilution) normalize technical variability across heat-shocked vs control samples.
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Subcellular compartmentalization: Nuclear-enriched protocols miss 68% of HSPA1A signal; whole-cell lysis with 0.1% NP-40 improves detection .
