HSP17.9A Antibody

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Description

Definition and Biological Context

HSP17.9A belongs to the small heat shock protein (sHSP) family, which stabilizes cellular proteins and membranes during stress conditions such as elevated temperatures . The HSP17.9A antibody is a polyclonal or monoclonal reagent developed to detect and quantify this protein in experimental settings, enabling researchers to study its expression patterns and functional roles .

Antibody Development and Validation

The HSP17.9A antibody has been validated across multiple plant species and experimental conditions:

ParameterDetails
Host SpeciesRabbit (polyclonal) , Mouse (monoclonal analogs for related sHSPs)
Target ReactivityPlant-specific (e.g., maize, grapevine)
ApplicationsWestern blot (WB), Immunofluorescence (IF), qPCR validation
Detection Sensitivity1:4000–1:100,000 dilution range

Key validation steps include:

  • Western Blot: Clear detection of ~17–27 kDa bands in heat-stressed plant tissues .

  • Expression Profiling: Correlation with qPCR data confirming upregulation under heat stress .

Heat Stress Response in Plants

Studies using the HSP17.9A antibody revealed:

  • Expression Dynamics: HSP17.9A is upregulated during heat shock (HS), with peak expression observed in thermotolerant plant varieties. For example:

Plant VarietyHeat TreatmentRelative Expression (Fold Change)
Grapevine (TN)Severe HS (45°C)12.5×
Maize (G342)45°C for 4 hours9.8×
Maize (K1)45°C for 4 hours7.2×
Data derived from
  • Tissue-Specific Localization: In soybean nodules, homologs like GmHSP17.9 were localized to infected zones, where they stabilize sucrose synthase activity and nitrogenase function .

Mechanistic Insights

  • Chaperone Activity: HSP17.9A interacts with proteins like GmNOD100 in soybean, enhancing sucrose synthase activity and maintaining nodule function under stress .

  • Membrane Stabilization: Analogous sHSPs (e.g., Synechocystis HSP17) bind lipids to prevent membrane hyperfluidization during heat stress .

Experimental Protocols

  • Western Blot: Optimal results achieved with 25 µg protein/lane, using Tris-glycine gels and PVDF membranes .

  • Antibody Dilution: 1:4000 for polyclonal HSP17.9A ; 1:20,000–1:100,000 for monoclonal analogs .

Cross-Reactivity and Limitations

  • Species Specificity: Strong reactivity in maize and grapevine; limited data in non-plant systems .

  • Batch Variability: Commercial antibodies (e.g., Proteintech 66767-1-Ig) require validation for each application .

Implications for Agricultural Biotechnology

The HSP17.9A antibody has facilitated breakthroughs in:

  • Crop Improvement: Identifying heat-tolerant genotypes via HSP17.9A expression screening .

  • Stress Physiology: Deciphering molecular pathways linking HSP17.9A to photosynthetic efficiency and yield stability under climate stress .

Future Directions

  • Structural Studies: Resolving HSP17.9A’s oligomeric state and interaction networks.

  • Field Applications: Developing HSP17.9A-based biomarkers for rapid phenotyping of stress resilience.

Product Specs

Buffer
Preservative: 0.03% Proclin 300
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Form
Liquid
Lead Time
Made-to-order (14-16 weeks)
Synonyms
HSP17.9A antibody; RNB5 antibody; Os03g0266300 antibody; LOC_Os03g15960 antibody; OJ1364E02.617.9 kDa class I heat shock protein antibody; 17.9 kDa heat shock protein 1 antibody; OsHsp17.9A antibody
Target Names
HSP17.9A
Uniprot No.

Target Background

Database Links
Protein Families
Small heat shock protein (HSP20) family
Subcellular Location
Nucleus.

Q&A

What is HSP17.9A and why is it significant for research?

HSP17.9A is a small heat shock protein (sHSP) with a molecular weight of approximately 17.9 kDa. It belongs to a ubiquitous class of molecular chaperones that protect cells against various stresses. The significance of HSP17.9A lies in its dual functionality - it prevents irreversible protein denaturation by binding non-native proteins in an ATP-independent manner and can stabilize cellular membranes during stress conditions . In plants like soybean, homologs such as GmHSP17.9 are critical for nodule development and nitrogen fixation, making them essential targets for agricultural research . HSP17.9A expression is particularly dynamic during stress responses, making it an excellent molecular marker for stress adaptation studies .

How does HSP17.9A differ from other small heat shock proteins?

HSP17.9A differs from other sHSPs in several key aspects:

  • Expression patterns: HSP17.9A shows tissue-specific expression patterns. For example, in soybean, GmHSP17.9 is specifically expressed in infected regions of nodules during development .

  • Functional specificity: Unlike some sHSPs that function primarily as protein chaperones, HSP17.9A homologs like Synechocystis HSP17 demonstrate an amphitropic nature - they can both interact with proteins and stabilize membranes .

  • Substrate selectivity: Some sHSPs show substrate selectivity in their chaperone activity. For instance, HSP-17 from C. elegans exhibits opposing activities on different substrates, promoting aggregation of malate dehydrogenase while inhibiting aggregation of citrate synthase and glyceraldehyde-3-phosphate dehydrogenase .

  • Structural features: Sequence analysis of HSP17.9A homologs reveals a conserved α-crystallin domain characteristic of sHSPs, but with specific sequence variations that determine its unique functionality .

What criteria should be considered when selecting an HSP17.9A antibody for research?

When selecting an HSP17.9A antibody for research applications, consider these critical factors:

  • Species specificity: Ensure the antibody recognizes HSP17.9A from your species of interest. The epitope recognition can vary significantly between plant, bacterial, and animal HSP17.9A homologs .

  • Cross-reactivity profile: Validate whether the antibody cross-reacts with other sHSPs, as many share conserved domains. Antibodies should be tested against knockout lines or purified recombinant proteins to confirm specificity .

  • Application compatibility: Confirm the antibody works in your intended applications (Western blot, immunohistochemistry, ELISA, etc.). Many antibodies perform well in Western blot but fail in other applications .

  • Validation data: Review existing validation data including Western blot results showing appropriate molecular weight detection (~17-18 kDa), knockout validation, and immunohistochemistry patterns .

  • Clonality: Consider whether polyclonal or monoclonal antibodies better suit your research needs. Polyclonal antibodies often provide stronger signals but may have batch-to-batch variability .

Antibody TypeAdvantagesLimitationsBest Applications
PolyclonalHigher sensitivity, Recognizes multiple epitopesBatch variation, Potential cross-reactivityWestern blot, ELISA
MonoclonalConsistent performance, Higher specificityLower sensitivity, Limited epitope recognitionImmunohistochemistry, Flow cytometry

How can I validate the specificity of an HSP17.9A antibody?

Rigorous validation is essential before using an HSP17.9A antibody in critical experiments:

  • Western blot against recombinant protein: Express and purify recombinant HSP17.9A and confirm the antibody detects a band at the expected molecular weight (~17-18 kDa) .

  • Knockout/knockdown validation: Compare antibody reactivity in wild-type versus HSP17.9A knockout or knockdown samples. A specific antibody will show significantly reduced or absent signal in knockout samples, as demonstrated for human HSP27 antibody validation .

  • Preabsorption controls: Preincubate the antibody with purified antigen before immunostaining to confirm that this abolishes specific staining .

  • Cross-species reactivity: Test the antibody against HSP17.9A homologs from different species to determine cross-reactivity if working with multiple organisms .

  • Mass spectrometry correlation: Immunoprecipitate HSP17.9A using the antibody and confirm protein identity by mass spectrometry to validate antibody specificity .

What are the optimal protocols for detecting HSP17.9A expression during stress responses?

To effectively detect HSP17.9A expression during stress responses, consider these methodological approaches:

  • qPCR gene expression analysis:

    • Select appropriate reference genes such as UBC and VAG, which have been validated for HSP17.9A expression studies .

    • Use the relative expression ratio calculation: (E target)^ΔCq target(control-treatment) / (E reference)^ΔCq reference(control-treatment) .

    • Perform biological and technical replicates (n ≥ 2) with standard deviation less than 0.5 .

    • Validate results using statistical tests like one-way ANOVA and Fisher's protected least significant difference test .

  • Western blot detection:

    • Separate proteins using reducing conditions and appropriate separation systems (12-230 kDa) .

    • Load appropriate controls including positive control samples and knockout/knockdown samples .

    • Use optimized antibody dilutions (typically 0.1-0.5 μg/ml for purified antibodies) .

    • Include loading controls like GAPDH to normalize protein levels .

  • In situ hybridization:

    • Prepare tissue sections at different developmental or stress treatment stages .

    • Use both sense and antisense probes specific for HSP17.9A .

    • Include negative controls to confirm specificity of hybridization signals .

How should experiments be designed to investigate HSP17.9A's role in stress tolerance?

A comprehensive experimental design to study HSP17.9A's role in stress tolerance should include:

  • Expression manipulation approaches:

    • Generate overexpression lines using appropriate vectors and promoters .

    • Create RNAi knockdown or CRISPR-Cas9 knockout lines to examine loss-of-function effects .

    • Develop composite plants (if working with plants) with tissue-specific expression modifications .

  • Stress treatment regimens:

    • Design heat stress treatments that mimic natural conditions (e.g., gradual temperature increases rather than sudden shocks) .

    • Include acclimation phases (moderate stress, 32°C for 3h) followed by extreme stress (42°C for 1h) and recovery periods .

    • Monitor multiple timepoints during stress and recovery to capture dynamic responses .

  • Phenotypic and functional assessments:

    • Measure relevant physiological parameters (e.g., in plants: photosynthetic activity, enzyme activities) .

    • Quantify stress indicators such as reactive oxygen species (ROS) content and peroxidase activity .

    • For plant nodulation studies, assess nodule number, fresh weight, and nitrogenase activity using acetylene reduction assay .

  • Molecular interaction studies:

    • Identify protein interaction partners using pull-down assays with purified recombinant HSP17.9A .

    • Verify interactions using methods like yeast two-hybrid or co-immunoprecipitation .

    • Investigate how stress conditions affect these protein-protein interactions .

How can protein-protein interactions of HSP17.9A be reliably characterized?

Characterizing HSP17.9A protein interactions requires multiple complementary approaches:

  • Pull-down assays:

    • Express HSP17.9A as a fusion protein (e.g., His-HSP17.9A-Avi) and purify using appropriate resin .

    • Incubate with total proteins isolated from relevant tissues or expression systems .

    • Detect interactions via Western blot using antibodies against potential interacting proteins .

    • Include negative controls with unrelated proteins expressed with the same tag system .

  • Co-immunoprecipitation (Co-IP):

    • Use HSP17.9A antibodies to pull down native protein complexes from tissue lysates .

    • Identify interacting partners by Western blot or mass spectrometry .

    • Perform reciprocal Co-IPs to confirm interactions .

  • Yeast two-hybrid screening:

    • Use HSP17.9A as bait to screen cDNA libraries from relevant tissues .

    • Validate positive interactions with directed yeast two-hybrid assays .

    • Confirm interactions using in vitro or in vivo methods .

  • Bimolecular fluorescence complementation (BiFC):

    • Create fusion constructs of HSP17.9A and potential interactors with split fluorescent protein fragments .

    • Transiently express in appropriate cells to visualize interactions through fluorescence microscopy .

    • Include appropriate controls to rule out false positive interactions .

What approaches can resolve contradictory data regarding HSP17.9A's function?

When encountering contradictory data about HSP17.9A function, implement these resolution strategies:

  • Substrate-specific effects investigation:

    • Test multiple substrates to determine if HSP17.9A has different effects on different proteins, as observed with HSP-17 from C. elegans that promotes aggregation of MDH but inhibits aggregation of CS and GAPDH .

    • Use varying concentrations of HSP17.9A, as some sHSPs show concentration-dependent effects (e.g., holdase activity at low concentrations but aggregase activity at higher concentrations) .

  • Environmental condition variations:

    • Systematically test different temperatures, as HSP17.9A activity may vary between moderate (37°C) and severe (45°C) heat stress conditions .

    • Examine effects under different pH conditions and ionic strengths .

    • Investigate membrane interactions that might modulate HSP17.9A function .

  • Technical approach diversification:

    • Employ multiple technical approaches to study the same phenomenon (e.g., light scattering, SDS-PAGE analysis of soluble/insoluble fractions, functional enzyme assays) .

    • Analyze both in vitro reconstituted systems and in vivo cellular contexts .

  • Protein state and modification analysis:

    • Investigate if post-translational modifications affect HSP17.9A function .

    • Examine oligomerization states, as many sHSPs function as oligomers with dynamic subunit exchange .

    • Use techniques like native PAGE and size exclusion chromatography to assess oligomeric state .

How does the methodology for studying HSP17.9A differ between plant and microbial systems?

The methodology for studying HSP17.9A varies significantly between plant and microbial systems:

Plant Systems:

  • Transformation approaches: Plants require Agrobacterium-mediated transformation, particle bombardment, or generation of composite plants with transformed roots .

  • Gene editing: CRISPR-Cas9 systems for plants must be optimized for plant cell delivery and selection .

  • Tissue-specific analysis: In plants like soybean, tissue-specific expression in nodules requires specialized sampling techniques and RNA in situ hybridization .

  • Phenotypic assessment: Plant studies focus on nodulation parameters, nitrogen fixation (acetylene reduction assay), and stress tolerance phenotypes .

  • Tissue culture requirements: Plant systems may require in vitro culture to control environmental conditions precisely during stress experiments .

Microbial Systems:

  • Transformation efficiency: Microbial systems like cyanobacteria allow for simpler and more efficient transformation protocols .

  • Growth conditions: Cyanobacterial studies can manipulate growth conditions more precisely in liquid culture .

  • Membrane studies: Investigation of HSP17.9A interaction with thylakoid membranes in cyanobacteria requires specialized membrane isolation techniques .

  • Protein production: Recombinant HSP17.9A can be produced at higher yields in bacterial expression systems for biochemical studies .

  • Genetic manipulation: Creation of knockout mutants is typically more straightforward in microbial systems .

What are the most advanced applications of HSP17.9A antibodies in functional genomics research?

Advanced applications of HSP17.9A antibodies in functional genomics research include:

  • Chromatin immunoprecipitation sequencing (ChIP-seq):

    • When HSP17.9A interacts with transcription factors or chromatin, antibodies can help identify genome-wide binding sites .

    • This approach reveals how HSP17.9A influences gene expression networks during stress responses.

  • Proteome-wide interaction mapping:

    • Using HSP17.9A antibodies for immunoprecipitation followed by mass spectrometry (IP-MS) reveals the complete interactome .

    • This identifies all proteins that associate with HSP17.9A under different stress conditions.

  • In vivo dynamics using fluorescent antibodies:

    • Fluorescently labeled antibody fragments can track HSP17.9A localization and dynamics in living cells .

    • This reveals real-time redistribution during stress responses.

  • Single-cell analysis:

    • HSP17.9A antibodies enable single-cell profiling to understand cell-to-cell variation in stress responses .

    • This is particularly valuable for studying heterogeneous tissues like nodules.

  • Proximity-dependent labeling:

    • HSP17.9A fusion proteins with proximity labeling enzymes combined with specific antibodies identify transient interaction partners .

    • This reveals the dynamic protein neighborhood of HSP17.9A during stress.

  • CRISPR screening validation:

    • HSP17.9A antibodies validate phenotypes from CRISPR screens targeting HSP17.9A or its interacting partners .

    • This confirms protein-level changes correspond to genetic modifications.

How can researchers overcome specificity issues when using HSP17.9A antibodies?

Researchers can address HSP17.9A antibody specificity challenges through these strategies:

  • Epitope mapping and selection:

    • Choose antibodies raised against unique regions of HSP17.9A rather than conserved domains shared with other sHSPs .

    • Consider developing custom antibodies against unique peptide sequences if commercial options lack specificity .

  • Validation with genetic controls:

    • Always include appropriate knockout or knockdown samples as negative controls .

    • Use overexpression samples as positive controls to confirm signal specificity .

  • Pre-adsorption tests:

    • Pre-incubate antibodies with recombinant HSP17.9A protein to compete away specific binding .

    • Compare staining patterns with and without pre-adsorption to identify non-specific signals .

  • Multiple antibody approach:

    • Use multiple antibodies recognizing different epitopes of HSP17.9A to confirm results .

    • Consistent results across different antibodies increase confidence in specificity .

  • Western blot optimization:

    • Increase stringency of washing steps and blocking conditions to reduce non-specific binding .

    • Optimize antibody concentration to minimize background while maintaining specific signal .

  • Mass spectrometry validation:

    • Confirm the identity of immunoprecipitated proteins by mass spectrometry to verify antibody captures the intended target .

What methodological modifications are needed when HSP17.9A expression levels are very low?

When dealing with low HSP17.9A expression levels, implement these methodological enhancements:

  • Enhanced protein extraction:

    • Use optimized extraction buffers with appropriate detergents to improve solubilization .

    • Include protease inhibitors to prevent degradation during sample preparation .

    • Concentrate proteins using methods like TCA precipitation or immunoprecipitation before analysis .

  • Signal amplification techniques:

    • Implement tyramide signal amplification for immunohistochemistry to enhance detection sensitivity .

    • Use high-sensitivity ECL substrates for Western blot detection .

    • Consider biotin-streptavidin amplification systems for low-abundance proteins .

  • qPCR modifications:

    • Increase starting RNA amount when possible .

    • Use pre-amplification steps for low-abundance transcripts .

    • Select high-efficiency primers (>95% efficiency) for HSP17.9A detection .

    • Implement digital PCR for absolute quantification of very low copy numbers .

  • Sample enrichment approaches:

    • Use subcellular fractionation to enrich for compartments where HSP17.9A is localized .

    • Implement immunoprecipitation to concentrate HSP17.9A before analysis .

    • For tissue-specific expression, use laser capture microdissection to isolate relevant cells .

  • Alternative detection methods:

    • Consider using Simple Western automated capillary-based immunoassays that offer higher sensitivity than traditional Western blots .

    • Implement proximity ligation assay (PLA) for detecting protein interactions at very low expression levels .

What emerging technologies will advance HSP17.9A research beyond current limitations?

Emerging technologies poised to transform HSP17.9A research include:

  • Single-molecule imaging techniques:

    • Super-resolution microscopy will enable visualization of HSP17.9A dynamics and interactions at nanometer resolution .

    • Single-molecule tracking can reveal the kinetics of HSP17.9A interactions with client proteins and membranes in living cells .

  • CRISPR-based approaches:

    • CRISPRi and CRISPRa will enable precise temporal control of HSP17.9A expression without permanent genetic modification .

    • Base editing and prime editing will allow introduction of specific mutations to study structure-function relationships .

  • Protein structure determination:

    • Cryo-electron microscopy will reveal the structure of HSP17.9A oligomers and their complexes with client proteins .

    • AlphaFold and other AI-based structural prediction tools will provide insights into HSP17.9A conformational dynamics .

  • Nanobody and aptamer development:

    • HSP17.9A-specific nanobodies will enable in vivo tracking with minimal interference to function .

    • RNA aptamers against HSP17.9A will provide alternative detection methods and potential regulatory tools .

  • Single-cell multiomics:

    • Integration of transcriptomics, proteomics, and metabolomics at single-cell resolution will reveal cell-type-specific roles of HSP17.9A .

    • Spatial transcriptomics will map HSP17.9A expression patterns with unprecedented resolution .

  • Organoid and tissue-on-chip models:

    • Advanced 3D culture systems will enable study of HSP17.9A function in more physiologically relevant contexts .

    • Microfluidic systems will allow precise control of stress conditions while monitoring HSP17.9A dynamics .

How can researchers integrate HSP17.9A functional data across different experimental systems?

To effectively integrate HSP17.9A functional data across different experimental systems, researchers should:

  • Develop standardized assays:

    • Establish common functional assays that can be applied across systems (e.g., standardized chaperone activity assays) .

    • Create benchmark datasets using reference strains or cell lines to enable cross-laboratory comparisons .

  • Implement computational integration approaches:

    • Utilize network analysis to identify conserved interaction partners across different species .

    • Develop machine learning models that can predict HSP17.9A function based on sequence and structural features .

  • Create comprehensive databases:

    • Establish repositories for HSP17.9A functional data that include standardized metadata about experimental conditions .

    • Implement ontologies to facilitate data comparison across different experimental systems .

  • Conduct comparative studies:

    • Directly compare HSP17.9A from different species in identical experimental setups .

    • Express HSP17.9A from one species in heterologous systems to assess functional conservation .

  • Utilize evolutionary approaches:

    • Analyze HSP17.9A sequence and functional conservation across evolutionary diverse organisms .

    • Identify core conserved functions versus species-specific adaptations .

  • Implement integrative modeling:

    • Develop mathematical models that integrate data from different levels of organization (molecular, cellular, organismal) .

    • Use systems biology approaches to predict emergent properties of HSP17.9A function in complex systems .

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