HSFA1B Antibody
Description
Introduction to HSFA1B Antibody
HSFA1B antibody is a specialized immunological reagent designed to specifically detect and bind to Heat Shock Factor Protein 1 (HSF1), also referred to as HSFA1B in some research contexts. These antibodies are purified immunoglobulins that have been validated for both specificity and sensitivity in detecting their target protein . Available as polyclonal primary antibodies, they serve as essential tools in research laboratories focused on understanding transcriptional regulation, heat shock responses, and cellular adaptation to environmental stresses.
The development of reliable HSFA1B antibodies has significantly contributed to our understanding of how organisms respond to thermal stress at the molecular level. These antibodies enable researchers to track the expression, localization, and activity of HSFA1B protein across various experimental conditions, thereby elucidating its role in stress-responsive gene networks.
Antibody Characteristics
The HSFA1B antibody is typically available as a polyclonal primary antibody, derived from rabbit host systems immunized with human HSF1 protein . These antibodies are presented in IgG format, reflecting their structural classification and functional properties. The immunogen used for antibody production typically consists of human HSF1 (amino acids 1-529) expressed in E. coli systems, ensuring recognition of the full-length protein .
Physical and Chemical Properties
| Property | Specification |
|---|---|
| Format | IgG |
| Size | 150 μg |
| Purity | Affinity Purified |
| Presentation | PBS pH 7.4, 50% Glycerol, 0.02% Sodium Azide |
| Storage Conditions | -20°C or below |
| Host | Rabbit |
| Antibody Type | Polyclonal |
| Isotype | Not specified |
| Conjugate Type | None |
Table 1: Physical and chemical properties of HSFA1B antibody .
Target Protein Information
The target of HSFA1B antibody is a protein involved in transcriptional regulation processes. Key identifiers for this target include:
| Identifier | Information |
|---|---|
| Entrez Gene | 3297 |
| UniProt | Q00613 |
| Synonyms | Heat Shock Transcription Factor 1, HSTF 1, Heat Shock Protein 1, HSF1 |
| Research Area | Transcription, Translation |
Table 2: Target protein identifiers for HSFA1B antibody .
Biological Role of HSFA1B
Understanding the biological role of HSFA1B is essential for appreciating the significance of HSFA1B antibodies in research applications.
HSFA1B as a Transcriptional Regulator
HSFA1B functions as a transcription factor that plays a critical role in regulating gene expression, particularly in response to heat stress. Research has demonstrated that HSFA1B can directly bind to specific DNA sequences known as heat shock elements (HSEs) in the promoters of target genes . This binding activity is fundamental to the protein's role in activating stress-responsive gene expression programs.
In Arabidopsis thaliana, HSFA1B overexpression has been shown to significantly alter the expression of over 500 genes under non-stress conditions . Transcriptomic analyses have revealed that many of these genes are associated with responses to both biotic and abiotic stresses, highlighting the multifaceted regulatory role of HSFA1B in stress adaptation mechanisms.
HSFA1B in Heat Stress Response
HSFA1B is a central component of the cellular heat stress response. In plants, HSFA1-type transcription factors often act redundantly to coordinate responses to elevated temperatures . The heat shock response mediated by HSFA1B involves the activation of genes encoding heat shock proteins (HSPs), which function as molecular chaperones to prevent protein misfolding and aggregation under thermal stress.
Research has demonstrated that HSFA1B can bind to specific heat shock elements (HSEs) in the promoters of genes such as HSP70.3 and HSP90.1 . These HSEs typically conform to the consensus sequence 5′-nGAAnnTTCnnGAAn-3′ . The binding of HSFA1B to these elements initiates transcriptional activation of the associated genes, thereby promoting cellular thermotolerance.
HSFA1B in Disease Resistance
Intriguingly, HSFA1B appears to have functions beyond heat stress response. Research with Arabidopsis plants overexpressing HSFA1B (HSFA1bOx) has demonstrated enhanced resistance to pathogens such as Hyaloperonospora parasitica (Hpa) and Pseudomonas syringae (Pst) . Conversely, plants with mutations in both HSFA1a and HSFA1b (hsfA1a/hsfA1b double mutants) showed increased susceptibility to these pathogens .
This role in disease resistance appears to be mediated through hydrogen peroxide (H₂O₂) signaling rather than through conventional stress hormone pathways involving salicylic acid (SA), jasmonic acid (JA), or abscisic acid (ABA) . Plants overexpressing HSFA1B showed elevated levels of H₂O₂, which may contribute to their enhanced immunity against pathogen infection .
Research Applications of HSFA1B Antibody
HSFA1B antibodies serve as valuable tools across various research applications, enabling investigations into the expression, localization, and activity of this important transcription factor.
Immunoassay Applications
Commercial HSFA1B antibodies are validated for use in enzyme immunoassay (EIA) and radioimmunoassay (RIA) applications . These techniques allow researchers to quantitatively measure HSFA1B protein levels in biological samples, facilitating studies on its expression patterns under various experimental conditions.
Chromatin Immunoprecipitation Studies
A particularly important application of HSFA1B antibodies is in chromatin immunoprecipitation (ChIP) experiments. These assays allow researchers to identify genomic regions bound by HSFA1B in vivo, providing insights into its direct regulatory targets.
Research using HSFA1B antibodies in ChIP-PCR experiments has demonstrated that HSFA1B binds to specific promoter elements, such as the HSE1b motif, in plants . For example, in Arabidopsis plants overexpressing an HSFA1b-RFP fusion protein, ChIP-PCR confirmed the binding of HSFA1B to HSE1b elements in the promoters of various genes, including several transcription factors .
Protein Interaction Studies
HSFA1B antibodies can also be utilized in co-immunoprecipitation (co-IP) assays to investigate protein-protein interactions involving HSFA1B. Recent research has demonstrated that HSFA1 family members can interact with other transcription factors, such as BES1, to coordinate responses to heat stress .
In vitro pull-down assays and bimolecular fluorescence complementation (BiFC) experiments have provided evidence for direct interactions between HSFA1a and BES1 . Similar approaches using HSFA1B antibodies could elucidate specific interaction partners of HSFA1B, offering insights into its regulatory networks.
HSFA1B-Regulated Gene Networks
Research employing HSFA1B antibodies has contributed significantly to our understanding of the gene networks regulated by this transcription factor.
Transcriptional Targets of HSFA1B
Transcriptomic analyses of Arabidopsis plants overexpressing HSFA1B have identified numerous genes that show altered expression in response to elevated HSFA1B levels . Under non-stress conditions, HSFA1B overexpression affected the expression of 509 genes, with 352 showing upregulation (>2-fold) and 157 showing downregulation (<0.5-fold) .
Many of these differentially expressed genes were associated with responses to heat stress and pathogen infection, consistent with the observed phenotypes of HSFA1B-overexpressing plants . The significant overlap between HSFA1B-regulated genes and genes responsive to heat stress or pathogen infection is summarized in the following table:
| Stress Condition | Total DEGs from Stress Microarrays | Genes Present in HSFA1bOx1/Ws-2 Data Set | Overlapping DEGs | P-value |
|---|---|---|---|---|
| Heat | 815 | 397 | 161 | 1.4×10⁻¹³³ |
| Drought | 4407 | 397 | 94 | 0.003 |
| Pst Infection | 1314 | 397 | 124 | 6.5×10⁻⁶¹ |
| Hpa Infection | 224 | 147 | 33 | 7.7×10⁻³⁸ |
Table 3: Overlap between HSFA1B-regulated genes and stress-responsive genes in Arabidopsis .
Direct Regulation via HSE1b Elements
Chromatin immunoprecipitation experiments have identified specific genes directly regulated by HSFA1B through binding to HSE1b elements in their promoters . Among the 509 genes affected by HSFA1B overexpression, 55 were found to harbor a variant of the heat shock element (HSE) in their promoters, termed HSE1b .
Direct binding of HSFA1B to these HSE1b elements was confirmed for selected genes using ChIP-PCR, providing strong evidence for their direct regulation by HSFA1B . Notably, HSFA1B was shown to bind specifically to HSE1b elements but not to core HSE sequences lacking the consensus HSE1b sequence, highlighting the specificity of this transcription factor for particular DNA motifs .
Biochemical Interactions of HSFA1B
Understanding the biochemical interactions of HSFA1B provides insights into its regulatory mechanisms and functional significance.
DNA-Binding Properties
HSFA1B, like other heat shock transcription factors, demonstrates specific DNA-binding properties that are critical for its function in transcriptional regulation. In vitro DNA binding studies using electrophoretic mobility shift assays (EMSAs) have shown that HSFA1 family members can bind to HSE-containing fragments of gene promoters .
These binding interactions appear to be specific for HSE sequences, as demonstrated by competition assays with unlabeled competitor nucleotides. When unlabeled competitors with intact HSEs were included in binding reactions, they efficiently competed for binding to HSFA1, whereas mutated HSE versions did not . This specificity ensures precise targeting of heat-responsive genes.
Protein-Protein Interactions
HSFA1B functions within complex regulatory networks that involve interactions with other proteins. Recent research has demonstrated that HSFA1 family members can interact directly with other transcription factors, such as BES1, to coordinate responses to heat stress .
These protein-protein interactions appear to enhance DNA-binding efficiency and transcriptional activation. For example, when BES1 and HSFA1a were combined in DNA-binding reactions, they bound to HSE-containing promoter fragments with very high efficiency, suggesting cooperative activity .
Such interactions highlight the interconnected nature of stress response networks and emphasize the potential roles of HSFA1B in integrating various signaling pathways to coordinate appropriate cellular responses to environmental challenges.
Product Specs
Target Background
Q&A
How do I validate the specificity of an HSF1 antibody in my experimental model?
Methodological Answer:
Validation requires a multi-step approach:
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Knockout Controls: Use HSF1-knockout cell lysates in Western blotting (WB) to confirm absence of signal at 82 kDa .
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Cross-Reactivity Checks: Verify antibody reactivity across species (human, mouse, rat, monkey) using lysates from transfected cell lines .
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Application-Specific Validation: For flow cytometry (FCM), compare staining intensity in heat-shocked (42°C, 1 hr) vs. unstressed cells, as HSF1 translocates to the nucleus under stress .
Table 1: Example Validation Data for HSF1 Antibody #4356 (Cell Signaling Technology)
| Application | Dilution | Expected Result | Validation Metric |
|---|---|---|---|
| Western Blotting | 1:1000 | Single band at 82 kDa in human/mouse lysates | ≥90% signal reduction in knockout lysates |
| Flow Cytometry | 1:50 | Nuclear localization post-heat shock | ≥5-fold signal increase in stressed cells |
| Chromatin IP | 1:50 | Enrichment of HSP70 promoter region | ≥10-fold enrichment vs. IgG control |
What experimental factors influence HSF1 antibody performance in immunofluorescence (IF)?
Methodological Answer:
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Fixation Method: Methanol fixation preserves HSF1’s nuclear localization better than paraformaldehyde .
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Antigen Retrieval: For paraffin-embedded tissues, citrate buffer (pH 6.0) at 95°C for 20 min improves epitope accessibility .
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Stress Induction: Pre-treat cells with 17-AAG (10 μM, 4 hr) to inhibit HSP90, forcing HSF1 activation and nuclear accumulation .
How can I resolve contradictions in HSF1 subcellular localization data across studies?
Methodological Answer:
Conflicting localization reports often arise from:
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Fixation Artifacts: Methanol fixation may lyse nuclear membranes, creating false cytoplasmic signals. Validate with live-cell imaging using GFP-tagged HSF1.
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Stress Condition Variability: Ensure consistent heat shock duration (e.g., 1 hr at 42°C ± 0.5°C) across experiments .
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Antibody Clone Differences: Compare clones like [EP1710Y] (Abcam) vs. [D3L8I] (CST), which target distinct epitopes (C-terminal vs. N-terminal) .
Table 2: Antibody Clone Performance in Subcellular Localization
| Clone | Epitope Location | Nuclear Signal (Stressed Cells) | Cytoplasmic Signal (Unstressed) |
|---|---|---|---|
| E-4 (SCBT) | DNA-binding domain | Strong (95% cells) | Weak (≤10% cells) |
| EP1710Y | Regulatory domain | Moderate (70% cells) | Moderate (30% cells) |
What strategies enhance signal-to-noise ratios in HSF1 chromatin immunoprecipitation (ChIP)?
Methodological Answer:
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Crosslinking Optimization: Use dual crosslinking (1% formaldehyde + 2 mM DSG) to stabilize HSF1-DNA interactions .
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Sonication Parameters: Fragment chromatin to 200–500 bp; oversonication disrupts HSF1 binding sites near HSP70 promoters.
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Antibody Validation: Pre-test antibodies in ChIP-qPCR using known HSF1 targets (e.g., HSPA1A promoter) .
How do phosphorylation-specific HSF1 antibodies (e.g., anti-pSer326) improve mechanistic studies?
Methodological Answer:
Phospho-Ser326 antibodies (e.g., BosterBio #PB-1000) enable detection of activated HSF1:
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Kinase Inhibition: Treat cells with U0126 (MEK inhibitor) to reduce Ser326 phosphorylation and confirm antibody specificity .
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Time-Course Experiments: Monitor pSer326 levels at 0, 15, 30, and 60 min post-heat shock to correlate phosphorylation with HSP gene expression.
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Mutagenesis Controls: Use HEK293 cells transfected with HSF1-S326A mutant to validate phospho-antibody specificity .
