HSP17.6 Antibody

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

HSP17.6 belongs to a family of class I small heat shock proteins that are induced when plant cells experience stress conditions . This 17.6 kDa protein (UniProt: P13853, TAIR: At1g53540) serves as an important marker for cellular stress responses in Arabidopsis thaliana and other plant species . Understanding HSP17.6 expression and regulation provides insights into plant adaptation mechanisms to environmental stressors such as heat, drought, and oxidative damage. As a cytosolic protein that responds rapidly to stress conditions, HSP17.6 represents a valuable molecular tool for investigating plant cellular protection systems and stress signaling pathways.

What are the key specifications of commercially available HSP17.6 antibodies?

HSP17.6 antibodies are typically available as polyclonal antibodies raised in chicken (IgY) or rabbit hosts . These antibodies are specifically designed to detect the 17.6 kDa heat shock protein from Arabidopsis thaliana, with potential cross-reactivity in related plant species. Key specifications include:

• Immunogen: Recombinant protein derived from Arabidopsis thaliana HSP17.6 Ci (class one)

• Host: Typically chicken (hen) or rabbit

• Clonality: Polyclonal

• Format: Liquid, purified total IgY in PBS pH 8.0 with 0.02% sodium azide

• Application: Western Blotting (WB) at recommended dilution of 1:1000

• Confirmed reactivity: Arabidopsis thaliana (with potential reactivity in Iris and Pinus sylvestris)

• Expected molecular weight: 17.6 kDa

How should HSP17.6 antibodies be stored and handled for optimal performance?

For maximum antibody stability and performance, HSP17.6 antibodies should be stored at 4°C . To prevent degradation from repeated freeze-thaw cycles, it is recommended to make aliquots rather than repeatedly using the stock solution . Before opening the tubes, briefly spin them to ensure no liquid is adhering to the cap or sides, which could lead to sample loss . The antibody is typically preserved with 0.02% sodium azide, which is a hazardous substance requiring appropriate handling precautions by trained personnel . Proper storage and handling significantly affect experimental reproducibility and sensitivity in Western blot applications.

What are the optimal Western blotting conditions for HSP17.6 antibody?

For optimal Western blot results with HSP17.6 antibody, follow this validated protocol:

• Sample preparation: Extract total protein from plant tissue (typically 10 μg is sufficient) .

• Separation: Use 15% SDS-PAGE gels for optimal resolution of the 17.6 kDa protein .

• Transfer: Blot proteins to nitrocellulose membrane for approximately 1 hour .

• Blocking: Block with appropriate blocking buffer to reduce background.

• Primary antibody: Incubate with HSP17.6 antibody at a 1:1000 dilution for 1 hour at room temperature with agitation .

• Secondary antibody: Use appropriate HRP-conjugated secondary antibody (1:10,000 dilution) .

• Detection: Visualize using standard ECL (Enhanced Chemiluminescence) detection methods .

This protocol has been validated in studies examining HSP17.6 expression in stress versus non-stress conditions, where clear differences in protein levels are observable between stressed and non-stressed plant samples .

How can I verify stress-induced HSP17.6 expression in Arabidopsis?

Verification of stress-induced HSP17.6 expression requires careful experimental design with appropriate controls:

• Stress treatment setup: Subject Arabidopsis plants to controlled heat stress (38°C is commonly used) while maintaining non-stressed plants as controls .

• Timing considerations: Monitor expression at different time points, as HSP17.6 induction is typically rapid but transient.

• Protein extraction: Extract total protein from both stressed and non-stressed tissues using the same protocol to ensure comparability.

• Western blotting: Follow the optimized Western blotting procedure described above, loading equal amounts of protein from stressed and non-stressed samples.

• Quantification: Use densitometry software to quantify band intensity differences between stressed and control samples.

• Validation: Consider complementing protein expression data with qRT-PCR analysis of HSP17.6 transcript levels.

Published experiments demonstrate that HSP17.6 is barely detectable in non-stressed Arabidopsis samples but shows strong induction after heat stress treatment , making it an excellent marker for verifying successful stress treatment protocols.

How can I troubleshoot weak or absent signal when using HSP17.6 antibody?

When experiencing weak or absent signals with HSP17.6 antibody, consider these methodological solutions:

• Antibody concentration: Increase primary antibody concentration from 1:1000 to 1:500 if signal is weak.

• Sample quantity: Increase total protein loading to 15-20 μg if 10 μg doesn't yield detectable signal.

• Stress conditions: Verify that stress conditions are sufficient to induce HSP17.6 expression; temperature, duration, and recovery time significantly affect expression levels.

• Protein extraction method: Optimize extraction buffer to ensure efficient recovery of cytosolic proteins like HSP17.6.

• Membrane type: Nitrocellulose membranes are recommended for optimal antibody binding .

• Detection sensitivity: Use higher sensitivity ECL reagents if standard ECL gives insufficient signal .

• Antibody quality: If using aliquoted antibody, ensure it hasn't been subjected to repeated freeze-thaw cycles, which can reduce activity .

If problems persist, running a positive control (protein extract from heat-stressed Arabidopsis) alongside experimental samples can help distinguish between technical issues and biological absence of the target protein.

What are the potential cross-reactivity issues with HSP17.6 antibody?

Understanding potential cross-reactivity is crucial for accurate data interpretation:

• Confirmed reactivity: HSP17.6 antibodies are validated for Arabidopsis thaliana, with reported reactivity in Iris and Pinus sylvestris .

• Other plant species: When working with species other than Arabidopsis, preliminary validation is essential, as heat shock protein families share sequence homology across species.

• Related HSPs: Arabidopsis contains multiple small HSP family members with similar molecular weights. To ensure specificity when multiple bands appear, consider:

  • Running recombinant HSP17.6 as a positive control to identify the correct band

  • Comparing expression patterns in stressed vs. non-stressed samples (genuine HSP17.6 should show stress induction)

  • Using T-DNA insertion mutants or RNAi lines as negative controls to confirm band identity

• Specificity verification: If working with complex samples, consider performing immunoprecipitation followed by mass spectrometry to confirm antibody specificity.

How can HSP17.6 antibody be used to study protein-protein interactions?

HSP17.6 antibodies can be valuable tools for investigating protein-protein interactions in stress response mechanisms:

• Co-immunoprecipitation (Co-IP):

  • Use HSP17.6 antibody for immunoprecipitation from plant extracts

  • Analyze precipitated complexes by Western blotting for potential interacting partners

  • Ensure proper controls are included (non-stressed samples, pre-immune serum IP)

• Proximity-dependent labeling:

  • Combine with techniques like BioID or APEX to identify proteins in proximity to HSP17.6 during stress response

  • Use the antibody to confirm successful expression of fusion proteins

• Immunofluorescence microscopy:

  • While the product information primarily lists Western blotting applications , optimized protocols may allow for immunofluorescence detection

  • This can reveal subcellular localization changes of HSP17.6 during stress conditions

• Pull-down validation:

  • After identifying potential interactors through other methods, use HSP17.6 antibody to validate interactions by Western blotting

These methods can provide insights into how HSP17.6 functions within chaperone complexes during cellular stress responses.

How can I use HSP17.6 antibody to compare stress responses across different mutant lines?

Comparative analysis of stress responses using HSP17.6 antibody requires careful experimental design:

• Experimental setup:

  • Grow wild-type and mutant plants under identical conditions

  • Apply standardized stress treatments (e.g., 38°C heat treatment) for consistent durations

  • Include non-stressed controls for each genotype

• Sampling timeline:

  • Collect samples at multiple time points during stress treatment and recovery

  • This captures potential differences in induction kinetics between genotypes

• Quantitative Western blotting:

  • Load equal amounts of total protein from each sample

  • Include internal loading controls (e.g., actin or tubulin)

  • Use the HSP17.6 antibody at consistent dilution (1:1000)

  • Employ quantitative detection methods (fluorescent secondary antibodies or carefully controlled ECL)

• Data analysis:

  • Normalize HSP17.6 band intensity to loading controls

  • Compare fold-induction relative to non-stressed samples for each genotype

  • Apply appropriate statistical tests to determine significance of differences

This approach has been successfully used to identify regulatory factors affecting HSP expression during stress, as demonstrated in studies examining T-DNA insertion mutants and RNAi transgenic lines .

How does the reproducibility of HSP17.6 antibody compare to other stress marker antibodies?

Antibody reproducibility is critical for reliable research outcomes. While specific reproducibility data for HSP17.6 antibody is limited in the provided sources, we can draw insights from reproducibility studies of similar heat shock protein antibodies:

• Reproducibility metrics:

  • Antibody reproducibility is typically assessed using coefficient of variation (CV) between repeat measurements

  • Good reproducibility is generally indicated by CV values < 0.20

  • HSP60 antibodies (another heat shock protein family) have shown reproducibility with CV values of approximately 0.17

• Factors affecting reproducibility:

  • Batch-to-batch variation in antibody production

  • Sample preparation consistency

  • Western blotting technique standardization

  • Image acquisition and quantification methods

• Reproducibility enhancement:

  • Use the same antibody lot for comparative experiments

  • Include standard samples across multiple blots for inter-blot comparison

  • Implement rigorous protocol standardization

  • Validate key findings with independent antibody preparations

Researchers should conduct preliminary reproducibility assessments when beginning new projects with HSP17.6 antibody, particularly when quantitative comparisons are essential to the experimental design.

Can HSP17.6 antibody be used for immunohistochemistry in plant tissues?

While the product information primarily specifies Western blotting applications , researchers interested in immunohistochemistry (IHC) with HSP17.6 antibody should consider these methodological adaptations:

• Tissue preparation:

  • Fix plant tissues in paraformaldehyde (typically 4%)

  • Perform paraffin embedding or prepare cryosections depending on experimental needs

  • Include antigen retrieval steps to overcome fixation-induced epitope masking

• Antibody optimization:

  • Start with higher concentrations than used for Western blotting (1:100 to 1:500)

  • Extend incubation times (overnight at 4°C)

  • Test multiple blocking reagents to minimize background

• Controls:

  • Include tissues from non-stressed plants as negative controls

  • Use tissues from HSP17.6 knockout mutants as specificity controls

  • Consider pre-absorption controls with recombinant HSP17.6 protein

• Detection system:

  • Use fluorescent secondary antibodies for better quantification

  • Implement counterstaining to visualize cellular structures

While this application requires validation, successful adaptation of Western blot antibodies for IHC has precedent in plant research and could provide valuable spatial information about HSP17.6 expression during stress responses.

How can I develop quantitative ELISA assays using HSP17.6 antibody?

Developing a quantitative ELISA for HSP17.6 requires methodical adaptation of the Western blot antibody:

• ELISA format selection:

  • Sandwich ELISA: Requires two antibodies recognizing different epitopes

  • Indirect ELISA: Simpler but may have higher background

  • Competitive ELISA: Useful for small proteins like HSP17.6

• Protocol development:

  • Coating concentration: Test ranges from 1-10 μg/ml of capture antibody

  • Blocking: Optimize to minimize background while preserving specific signal

  • Sample preparation: Develop extraction protocols that preserve native protein conformation

  • Standard curve: Generate using recombinant HSP17.6 protein

  • Detection: HRP-conjugated secondary antibodies with colorimetric or chemiluminescent substrates

• Validation:

  • Determine specificity using HSP17.6 knockout or knockdown plant materials

  • Assess sensitivity by measuring detection limits with purified protein

  • Evaluate reproducibility through multiple independent experiments

  • Calculate coefficient of variation (CV) to ensure values < 0.20 for reliable quantification

• Applications:

  • High-throughput screening of stress responses across multiple samples

  • Quantitative comparison of HSP17.6 induction across different stress conditions

  • Analysis of HSP17.6 expression in developmental time courses

While developing a new ELISA requires significant optimization, the high specificity of available HSP17.6 antibodies provides a solid foundation for assay development.