HSP17.6C Antibody

What is HSP17.6C and why is it important in plant stress research?

HSP17.6C (17.6 kDa class I heat shock protein 3) belongs to the HSP20-like chaperones superfamily and is located in the cytoplasm. It plays a crucial role in the plant's response to heat stress by functioning as a molecular chaperone, protecting other proteins from irreversible denaturation under elevated temperatures . HSP17.6C is particularly significant in research because:

• It shows dramatic induction in response to heat stress conditions

• Its expression levels correlate strongly with heat stress severity

• It contributes to thermotolerance in plants such as Arabidopsis thaliana

• It is involved in epigenetic memory of heat stress through modified histone regulation

Studies have demonstrated that HSP17.6C is absent under normal growth conditions but becomes significantly expressed during heat stress, making it an excellent marker for investigating heat stress responses .

What are the optimal experimental conditions for Western blot detection of HSP17.6C?

For optimal Western blot detection of HSP17.6C, researchers should follow these methodological guidelines:

Sample preparation:

• Use 15-25 μg of total protein from heat-stressed plant tissue (higher amounts may be needed for control samples where expression is minimal)

• Include both heat-shocked and control plant samples for comparison

• Consider including purified recombinant HSP17.6C (1-10 ng) as a positive control

Electrophoresis and blotting conditions:

• Use 15% SDS-PAGE for optimal separation of low molecular weight proteins

• Transfer to nitrocellulose membrane for 1 hour

• Use 5% skim milk for blocking (at room temperature or 4°C for 1 hour)

Antibody application:

• Primary antibody dilution: 1:1000-1:2000 for Western blot applications

• Incubate with primary antibody overnight at 4°C or 1 hour at room temperature

• Secondary antibody (HRP-conjugated): 1:10,000 dilution

• Detection using chemiluminescence substrates

Example application protocol from validated studies:

"15 μg of total protein from heat-shocked Arabidopsis thaliana, control Arabidopsis thaliana plants, and 1,2,5,10 ng of recombinant purified HSP17.6 were separated on 15% SDS-PAGE and blotted for 1h to nitrocellulose. Blots were incubated in the primary antibody at a dilution of 1:1000 for 1h at room temperature with agitation and secondary HRP-conjugated antibody (1:10,000)."

Which plant species show confirmed cross-reactivity with HSP17.6C antibodies?

HSP17.6C antibodies have been tested and confirmed to react with heat shock proteins from multiple plant species. The cross-reactivity depends on the specific antibody used:

For antibody AS07 254 (targeting Arabidopsis thaliana HSP17.6 CI):

• Arabidopsis thaliana (primary target)

• Agave tequilana var. Weber

• Brassica juncea

• Citrus sp.

• Cucumis sativus

• Iris pumilla (perennial monocot)

• Pinellia ternata

• Pinus sylvestris

• Silene vulgaris

• Solanum tuberosum

• Vicia faba

For PHY0149S and PHY3850S antibodies:

• Arabidopsis thaliana

• Solanum tuberosum

• Medicago truncatula

• Additional predicted reactivity with: Triticum aestivum, Hordeum vulgare, Glycine max, and several other species

How does HSP17.6C expression correlate with different heat stress regimes, and how can this be accurately quantified?

Research has demonstrated that HSP17.6C expression shows a direct correlation with heat stress severity, with different patterns observed between gradual and abrupt heat treatments:

Expression patterns under different heat treatments:

• Control (no stress): HSP17.6C protein is virtually undetectable

• Gradual heat stress: Moderate HSP17.6C expression detected

• Abrupt heat stress: Significantly higher expression levels (approximately 6-fold more than gradual heat)

Relationship between mRNA poly(A) tail length and protein expression:

These findings suggest that the length of the poly(A) tail contributes significantly to translation efficiency and protein expression levels of HSP17.6C .

Quantification methodology:
For accurate quantification of HSP17.6C expression:

• Use densitometry software (e.g., Image Studio Lite) to measure band intensity

• Normalize against consistent loading controls such as Rubisco or actin

• Include both heat-shocked and control samples in the same blot

• For precise quantification, include a standard curve using recombinant HSP17.6C protein

What are the epigenetic mechanisms regulating HSP17.6C expression during heat acclimation?

Recent research has revealed sophisticated epigenetic mechanisms regulating HSP17.6C expression during heat acclimation and subsequent heat stress:

Histone modification dynamics:

• H3K27me3 (histone H3 lysine 27 trimethylation) acts as a repressive mark at the HSP17.6C locus under normal conditions

• During heat acclimation, JUMONJI (JMJ) demethylases remove H3K27me3 marks, creating an epigenetic memory of heat exposure

• This demethylation "primes" the HSP17.6C gene for rapid activation upon subsequent heat stress

• In jmjq mutants (lacking functional JMJ proteins), HSP17.6C expression is significantly reduced during heat stress after acclimation

ChIP-qPCR analysis results:
H3K27me3 levels at the HSP17.6C gene body decrease gradually following acclimation and heat stress in wild-type plants but remain elevated in jmjq mutants. This indicates that JMJ proteins are essential for modifying chromatin structure to facilitate HSP17.6C expression under stress conditions .

Methodological approach for investigating epigenetic regulation:

• Perform ChIP using antibodies against specific histone modifications (H3K27me3, H3K4me3)

• Quantify enrichment by qPCR using primers targeting the HSP17.6C gene body

• Compare wild-type plants with epigenetic modifier mutants (e.g., jmjq)

• Correlate histone modification levels with gene expression data from RT-qPCR or RNA-seq

These findings highlight the crucial role of epigenetic regulation in plant heat stress memory and acclimation .

How can I distinguish between different small heat shock proteins when antibody cross-reactivity is a concern?

Distinguishing between closely related small heat shock proteins can be challenging due to sequence homology and potential antibody cross-reactivity. Several strategic approaches can address this challenge:

Understanding antibody specificity:
The specificity of anti-HSP17.6C antibodies varies. For example, the synthetic peptide used for immunization of PHY3850S shares:

• 88% homology with HSP18.2 (AT5G59720) and HSP17.6B (AT2G29500)

• 81% homology with HSP17.4 (AT3G46230)

This high sequence similarity can lead to cross-reactivity with other class I small HSPs.

Methodological approaches to improve specificity:

• Genetic validation: Use knockout/knockdown mutants of specific HSPs as negative controls

• Recombinant protein controls: Include purified recombinant versions of different HSPs to identify cross-reactivity patterns

• Immunoprecipitation followed by mass spectrometry: Identify which specific HSPs are being detected by the antibody

• Alternative detection methods: Use transcript-specific approaches (qRT-PCR, RNA-seq) with highly specific primers

• Western blot optimization: Adjust antibody concentration and washing conditions to increase specificity

Example of distinguishing control methodology:
Researchers can use HSP12.6 as a control in experiments, as it has been reported to have distinct functional properties from HSP17.6, allowing clear differentiation between these small heat shock proteins .

What methods can be used to study the functional role of HSP17.6C in thermotolerance?

Investigating the functional significance of HSP17.6C in thermotolerance requires multiple complementary approaches:

Genetic manipulation approaches:

• Loss-of-function studies: Using hsp17.6c knockout/knockdown mutants to assess thermotolerance

  • Evidence shows that hsp22 hsp17.6c double mutants exhibit reduced heat acclimation capacity compared to wild-type plants

• Gain-of-function studies: Ectopic expression of HSP17.6C

  • Overexpression in jmjq mutants partially rescues heat-acclimation defects

• Complementation assays: Expressing HSP17.6C in knockout backgrounds to confirm specificity of observed phenotypes

Phenotypic assessment methods:

Biochemical and molecular approaches:

• Chaperone activity assays: Assess the ability of HSP17.6C to prevent aggregation of model substrate proteins

• Protein-protein interaction studies: Identify HSP17.6C binding partners using co-immunoprecipitation, yeast two-hybrid, or proximity labeling

• Subcellular localization: Determine where HSP17.6C functions during heat stress using fluorescent protein fusions

Experimental design for testing thermotolerance:

This multifaceted approach provides comprehensive insights into HSP17.6C function during heat stress response .

What are the optimal storage and handling conditions for HSP17.6C antibodies?

Proper storage and handling of HSP17.6C antibodies is critical for maintaining their activity and specificity. Based on manufacturer recommendations:

Storage conditions:

• Store lyophilized antibody at -20°C to -70°C

• After reconstitution, store at -20°C to -70°C for up to 6 months under sterile conditions

• For short-term storage (up to 1 month), store at 2-8°C under sterile conditions

Reconstitution protocol:

• For lyophilized antibody, add 50-150 μl of sterile water (depending on the specific product)

• Spin tubes briefly prior to opening to avoid any losses that might occur from material adhering to the cap or sides

• After reconstitution, make aliquots to avoid repeated freeze-thaw cycles

Handling precautions:

• Avoid repeated freeze-thaw cycles as they may lead to loss of antibody activity

• Before each use, thaw aliquots completely before use and mix gently

• Some products may contain preservatives like ProClin that should be noted in experimental protocols

Following these guidelines will help ensure consistent antibody performance across experiments and maximize the usable lifetime of the antibody.

What controls should be included when using HSP17.6C antibody for experimental validation?

Proper experimental controls are essential for validating HSP17.6C antibody specificity and ensuring reliable results:

Essential controls for Western blot experiments:

• Positive control: Include recombinant purified HSP17.6C protein (1-10 ng) to confirm antibody reactivity

• Negative control: Include samples from:

  • Non-heat-stressed plants (HSP17.6C should be minimal or absent)

  • When available, hsp17.6c knockout mutants to confirm antibody specificity

• Loading control: Use antibodies against housekeeping proteins like:

  • Rubisco (for plant samples)

  • Actin or tubulin (for general protein loading control)

  • The loading control should show consistent band intensity across samples

Control for cross-reactivity assessment:
When testing for potential cross-reactivity with other small HSPs, include recombinant versions of related proteins (HSP17.6B, HSP18.2, HSP17.4) to determine if the antibody detects these related proteins .

Controls for heat treatment experiments:

• Gradient of heat stress: Include samples from plants exposed to different temperatures or durations of heat stress to demonstrate dose-dependent expression

• Time course samples: Collect samples at multiple time points following heat stress to capture expression dynamics

• Recovery period samples: Include samples taken during recovery from heat stress to assess protein stability

Example of control implementation:

"15 μg of total protein from (HS) heat shocked Arabidopsis thaliana, (C) Arabidopsis thaliana control plants, and (1,2,5,10) 1,2,5,10 ng of recombinant purified HSP17.6 were separated on 15% SDS-PAGE... The band density of Rubisco on average was 765,000, 765,800, and 765,300, indicating that the loading protein was almost the same."

How can HSP17.6C antibody be used to study heat acclimation memory in plants?

HSP17.6C antibody serves as a powerful tool for investigating heat acclimation memory in plants, particularly when combined with other molecular approaches:

Experimental design for studying heat acclimation:

• Acclimation treatment: Expose plants to moderate heat stress (e.g., 37°C) followed by recovery period

• Memory testing: After recovery (3-7 days), expose plants to lethal heat stress

• Sample collection: Harvest tissue at key time points:

  • Before acclimation (control)

  • Immediately after acclimation

  • During recovery period

  • After subsequent heat stress

Molecular analyses using HSP17.6C antibody:

• Western blot: Quantify HSP17.6C protein levels across time points

• Immunolocalization: Determine cellular and subcellular distribution of HSP17.6C

• Chromatin immunoprecipitation (ChIP): Combine with antibodies against histone modifications to correlate with HSP17.6C expression levels

Integration with epigenetic studies:
Recent research has demonstrated that epigenetic mechanisms, particularly H3K27me3 demethylation by JUMONJI proteins, regulate HSP17.6C expression during heat acclimation. This creates a "primed" state that facilitates rapid HSP17.6C induction upon subsequent heat stress. Researchers can use HSP17.6C antibody to correlate protein expression with chromatin state changes .

Application to field conditions:
The study of HSP17.6C expression using specific antibodies can help understand how plants maintain heat memory under fluctuating field temperature conditions, which has significant implications for crop improvement in the context of climate change .

What is the relationship between HSP17.6C and other small heat shock proteins in conferring thermotolerance?

Understanding the functional relationships between HSP17.6C and other small heat shock proteins is crucial for comprehending the complete thermotolerance mechanism in plants:

Functional redundancy and specificity:

• Small HSPs often show overlapping yet distinct functions in stress response

• HSP17.6C belongs to the cytosolic class I small HSPs, which includes six genes in Arabidopsis thaliana

• HSP17.6C antibodies may cross-react with other class I small HSPs but not with class II, organelle, or other HSP classes

Cooperative interactions:
Research using genetic approaches has revealed that:

• HSP17.6C functions cooperatively with HSP22 in thermotolerance

• hsp22 hsp17.6c double mutants show more severe defects in heat acclimation than single mutants

• This suggests complementary yet partially overlapping functions

Methodological approaches to study interactions:

• Co-immunoprecipitation: Use HSP17.6C antibody to pull down protein complexes and identify interacting partners

• Sequential immunodepletion: Deplete specific HSPs from extracts to determine their relative contributions

• Genetic analysis: Compare phenotypes of single and higher-order mutants (as done with hsp22 hsp17.6c double mutants)

• Protein expression profiling: Use antibodies against multiple HSPs to profile their expression patterns under different stress conditions

Future research directions:

• Determining specific substrates protected by HSP17.6C versus other small HSPs

• Investigating potential functional hierachies among small HSPs during heat stress

• Exploring how specific combinations of small HSPs might be deployed in different plant tissues or in response to different stress intensities

Understanding these relationships will provide deeper insights into the molecular mechanisms of plant thermotolerance and inform strategies for improving crop resilience to heat stress .

How does mRNA processing affect HSP17.6C protein expression, and how can this be studied using HSP17.6C antibody?

Research has revealed a fascinating connection between mRNA poly(A) tail length and HSP17.6C protein expression levels, which can be investigated using HSP17.6C antibody in conjunction with molecular approaches:

Poly(A) tail length and translation efficiency:

• HSP17.6C transcripts exhibit variable poly(A) tail lengths depending on heat treatment conditions

• Longer poly(A) tails correlate with increased protein expression levels

• This relationship suggests post-transcriptional regulation plays a crucial role in heat stress response

Comparative data showing poly(A) tail length and protein expression:

This data demonstrates that poly(A) tail length elongation contributes significantly to increased HSP17.6C protein expression during heat stress .

Methodological approach for studying this relationship:

• APAL-seq (Assay for Poly(A) Tail Length - sequencing): Measure poly(A) tail length at the transcriptome level

• Western blot with HSP17.6C antibody: Quantify protein expression levels

• Correlation analysis: Compare poly(A) tail length data with protein expression data across different treatment conditions

• Genetic manipulation: Use mutants in poly(A) processing machinery to test the causal relationship

Research applications:

• Investigating how different types of heat stress affect mRNA processing and protein expression

• Exploring whether this mechanism applies to other heat shock proteins

• Understanding the temporal dynamics of poly(A) tail elongation and protein accumulation during stress response