HSP90-7 Antibody

What is HSP90.7 and how does it differ from other HSP90 isoforms?

HSP90.7 is one of seven HSP90 isoforms (HSP90.1-HSP90.7) found in Arabidopsis, specifically localized to the endoplasmic reticulum (ER) where protein folding actively occurs. Unlike cytoplasmic HSP90 variants, HSP90.7 belongs to the GRP94 protein family, which is characterized by specific structural features. A unique characteristic of plant-derived GRP94 proteins, including HSP90.7, is the presence of a short, charged region in the middle domain that is absent in animal GRP94 proteins . This charged region appears to be functionally significant, as deletion mutants (HSP90.7 Δ22) show enhanced sensitivity to ER stress induced by tunicamycin or high calcium concentrations, without significantly affecting general chaperone activity .

HSP90 proteins share a common structure consisting of an N-terminal ATP-binding domain, a substrate-binding middle domain, and a C-terminal dimerization domain . Despite structural similarities, functional studies have demonstrated that different HSP90 isoforms have distinct roles in cellular processes and cannot always compensate for each other, making isoform-specific antibodies crucial for research.

How should researchers validate the specificity of an HSP90.7 antibody?

Validation of HSP90.7 antibody specificity requires a multi-pronged approach to ensure accurate experimental results. First, Western blot analysis should be performed using both wild-type samples and knockout models. As demonstrated in HSP90α antibody validation, comparing parental cell lines with HSP90 knockout cell lines can confirm antibody specificity . A specific band at the expected molecular weight (approximately 90-96 kDa for HSP90 proteins) should be present in wild-type samples but absent in knockout samples .

Second, cross-reactivity testing against other HSP90 isoforms is essential. For example, R&D Systems' HSP90α antibody was validated by showing no cross-reactivity with recombinant human HSP90β in Western blots . For HSP90.7 antibodies, similar testing against HSP90.1-HSP90.6 proteins is necessary.

Third, researchers should validate antibody specificity using:

• Immunoprecipitation followed by mass spectrometry

• Immunofluorescence with appropriate controls

• Testing in multiple experimental systems where HSP90.7 expression has been manipulated

Loading controls (such as GAPDH) should always be included to ensure equal protein loading across samples . Additionally, researchers may confirm antibody reactivity against tagged versions of HSP90.7 (e.g., FLAG-tagged HSP90.7) as an additional validation step .

What are the recommended detection methods for HSP90.7 using antibodies?

HSP90.7 can be detected through several antibody-based methods, each with specific optimization requirements. Western blotting represents the most common detection method, requiring careful optimization of antibody concentration. For example, HSP90α antibodies have been successfully used at concentrations of 0.5 μg/mL with HRP-conjugated secondary antibodies . For HSP90.7, similar concentration ranges may be appropriate, but optimization is essential.

Immunofluorescence staining can visualize HSP90.7 cellular localization, particularly its ER association. Double-immunofluorescent staining may be useful for co-localization studies, as demonstrated in research examining HSP90 co-expression with cancer stem cell markers .

Flow cytometry can quantify HSP90.7 expression levels across cell populations. When developing flow cytometry protocols for HSP90.7, researchers should consider that as an ER protein, cell permeabilization will be necessary. This is distinct from the approach for detecting surface-expressed HSP90, as described in some cancer studies .

Co-immunoprecipitation using HSP90.7 antibodies can identify protein interaction partners. Successful co-IP protocols have been established using purified antibodies against HSP90, combined with SDS-PAGE and mass spectrometry for interactor identification .

How can HSP90.7 antibodies be utilized to study ER stress responses?

HSP90.7 antibodies serve as valuable tools for investigating ER stress responses, particularly in plant systems where HSP90.7 plays a critical role. When designing experiments to study ER stress using HSP90.7 antibodies, researchers should consider several methodological approaches:

First, time-course experiments examining HSP90.7 protein levels during ER stress induction provide insights into stress-responsive regulation. Researchers can induce ER stress using tunicamycin or high calcium concentrations (as demonstrated in HSP90.7 deletion mutant studies), then use Western blotting with HSP90.7 antibodies to track protein expression changes . When analyzing these experiments, it's important to distinguish between transcriptional and post-translational regulation, as HSP90.7 gene expression may not always correlate with protein levels, as observed in HSP90.7 knockout studies where the protein was absent despite minimal changes in transcript level .

Second, co-immunoprecipitation with HSP90.7 antibodies during normal and ER stress conditions can identify stress-specific interaction partners. This approach has been successfully used with other HSP90 isoforms and can be adapted for HSP90.7 . The resulting data should be analyzed using both qualitative (presence/absence of interactors) and quantitative (changes in interaction strength) parameters.

Third, subcellular fractionation followed by Western blotting can track HSP90.7 redistribution during ER stress. Changes in localization may indicate functional shifts in response to stress conditions.

What are the best approaches for studying HSP90.7 in relation to plant development and signaling pathways?

Studying HSP90.7's role in plant development requires integrating antibody-based techniques with developmental biology approaches. HSP90.7 knockout studies have revealed its importance in root meristem cell proliferation and expansion, with mutants showing reduced primary root growth and altered cell patterning . Researchers investigating HSP90.7 in development should:

First, combine HSP90.7 antibody staining with developmental markers to correlate protein expression with specific developmental stages. In Arabidopsis, researchers have successfully used markers like QC25GUS (quiescent center) and CYCB1-1GUS (cell division) alongside HSP90.7 analysis to understand its developmental function . Immunohistochemistry with HSP90.7 antibodies in tissue sections can provide spatial information about protein expression throughout development.

Second, study HSP90.7's interaction with developmental signaling networks using both genetic and biochemical approaches. Research has shown that HSP90.7 knockout affects auxin homeostasis, with mutants showing reduced PIN1 and PIN5 expression and downregulation of the TAA-YUCCA auxin biosynthesis pathway . Western blotting with HSP90.7 antibodies, combined with antibodies against signaling components (like β-catenin in cancer studies ), can reveal regulatory relationships.

Third, employ transgenic complementation experiments with epitope-tagged HSP90.7 variants that can be detected with commercial antibodies. This approach has been used to rescue HSP90.7 mutant phenotypes and confirm protein functionality .

How can HSP90.7 antibodies be used to elucidate chaperone-client relationships?

Elucidating HSP90.7's chaperone-client relationships requires methodologies that preserve transient interactions while providing sufficient specificity. Based on approaches used with other HSP90 isoforms, researchers should consider:

First, developing a comprehensive interactome using quantitative immunoprecipitation followed by mass spectrometry (IP-MS). This approach has successfully identified HSP90 client proteins in various systems . For HSP90.7, IP-MS should be performed under both normal and stress conditions, as client interactions may change in response to cellular stressors. When analyzing IP-MS data, researchers should distinguish between direct clients and indirect interactors by incorporating crosslinking approaches or proximity-based labeling.

Second, comparing client protein stability in the presence and absence of HSP90.7 can confirm functional chaperone-client relationships. After HSP90.7 knockdown or inhibition, Western blotting can assess client protein levels and processing. Studies with HSP90α demonstrated that modest knockdown affects client protein maturation, with flow cytometry showing reduced surface expression of receptor tyrosine kinases IGF-1R and HER2 after geldanamycin challenge in knockdown cells .

Third, examining ATP-dependent interactions can distinguish between different types of client relationships. Since HSP90.7 shows ATP-binding and hydrolysis activity , researchers can use ATP analogs or ATPase-deficient mutants to trap complexes at different stages of the chaperone cycle, then identify clients using HSP90.7 antibodies for immunoprecipitation.

What are the critical factors for optimizing Western blot protocols with HSP90.7 antibodies?

Optimizing Western blot protocols for HSP90.7 detection requires attention to several critical factors:

First, sample preparation significantly impacts results. Since HSP90.7 is an ER-localized protein, extraction buffers should effectively solubilize membrane-associated proteins. Complete lysis buffers containing 1-2% non-ionic detergents (such as Triton X-100) are recommended. Samples should be denatured at 95°C for 5 minutes in reducing sample buffer to ensure complete unfolding.

Second, antibody concentration requires careful titration. Starting with concentrations similar to those used for other HSP90 isoforms (e.g., 0.5 μg/mL as reported for HSP90α antibodies ) is recommended, followed by optimization. Blocking conditions significantly affect background and signal specificity; 5% non-fat dry milk in TBST is typically effective, but BSA-based blocking may be preferable for phospho-specific detection.

Third, detection system selection impacts sensitivity. While HRP-conjugated secondary antibodies provide good sensitivity for abundant proteins like HSP90.7, fluorescent secondary antibodies may offer advantages for multiplex detection and quantification. For example, simultaneous detection of HSP90.7 and other ER proteins or client proteins can be achieved with appropriate fluorescent secondary antibodies.

Fourth, proper controls are essential for interpretation. These include:

• Positive control (sample known to express HSP90.7)

• Negative control (HSP90.7 knockout/knockdown sample)

• Loading control (GAPDH is commonly used )

• Specificity control (recombinant HSP90 isoforms to test cross-reactivity )

How should researchers approach troubleshooting non-specific binding or weak signals with HSP90.7 antibodies?

When encountering non-specific binding or weak signals with HSP90.7 antibodies, researchers should implement a systematic troubleshooting approach:

For non-specific binding issues, first analyze the pattern of bands observed. Additional bands at unexpected molecular weights may indicate cross-reactivity with other HSP90 isoforms, degradation products, or non-specific binding. To address this:

• Increase antibody specificity by performing more stringent washing (higher salt concentration or mild detergent in wash buffer)

• Use alternative blocking agents (switch between milk and BSA)

• Perform pre-adsorption of the antibody with recombinant proteins of potential cross-reactive isoforms

• Reduce primary antibody concentration in small increments

• Use knockout or knockdown controls to confirm band identity

For weak signal issues, consider:

• Increasing protein loading (up to 50 μg per lane)

• Optimizing extraction to better solubilize ER-associated proteins

• Extending primary antibody incubation (overnight at 4°C)

• Using signal enhancement systems (such as biotin-streptavidin amplification)

• Checking for protein degradation during sample preparation

• Testing alternative antibodies recognizing different epitopes of HSP90.7

If problems persist, consider validation with alternative detection methods. For example, if Western blot continues to show weak signal, immunoprecipitation followed by mass spectrometry can confirm HSP90.7 presence and abundance .

What considerations are important when using HSP90.7 antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) with HSP90.7 antibodies presents unique challenges due to the protein's ER localization and chaperone function. Successful Co-IP requires:

First, lysis buffer optimization is critical. Harsh detergents may disrupt protein-protein interactions, while insufficient solubilization may leave HSP90.7 complexes in the insoluble fraction. A balanced approach using mild detergents (0.5-1% NP-40 or Triton X-100) with physiological salt concentrations (150 mM NaCl) often preserves interactions while achieving adequate solubilization. Protease inhibitors are essential to prevent degradation during extraction.

Second, antibody coupling strategy significantly impacts results. Direct coupling of HSP90.7 antibodies to beads (such as agarose G ) prior to immunoprecipitation can reduce background from heavy and light chains in subsequent analysis. Alternatively, using antibodies raised in species different from secondary detection antibodies allows differentiation between antibody chains and precipitated proteins.

Third, washing conditions must balance removing non-specific interactions while preserving specific ones. A step-wise washing approach with decreasing detergent concentrations often yields optimal results. For HSP90.7, which may form both stable and transient interactions, comparing multiple washing conditions in parallel experiments can help differentiate between these interaction types.

Fourth, elution strategy selection depends on downstream applications. For mass spectrometry analysis, elution with SDS sample buffer provides complete recovery but introduces contaminants. For functional studies of co-precipitated proteins, milder elution with peptide competition or pH changes may better preserve activity.

When analyzing Co-IP results, researchers should consider HSP90.7's role as a chaperone, which may result in interactions with unfolded proteins that are not physiological clients. Validation of potential interactions through reciprocal Co-IP, proximity ligation assays, or functional studies is strongly recommended.

How should researchers interpret changes in HSP90.7 expression levels across different experimental conditions?

Interpreting changes in HSP90.7 expression requires consideration of both biological and technical factors that may influence results:

First, distinguish between transcriptional and post-translational regulation. RNA-seq data from HSP90.7 knockout studies revealed that HSP90.7 gene expression was not significantly altered at the transcriptional level despite clear protein-level disruption . Therefore, researchers should combine protein expression analysis (using HSP90.7 antibodies) with transcriptional analysis (RT-qPCR or RNA-seq) to comprehensively understand regulation.

Second, consider the relationship between HSP90.7 and stress responses. As an ER chaperone, HSP90.7 may respond differently to various stressors. For example, HSP90.7 deletion mutants show enhanced sensitivity to ER stress induced by tunicamycin or high calcium , suggesting functional importance under these conditions. When interpreting stress-related expression changes, researchers should:

• Establish time-course profiles to distinguish between early and late responses

• Compare HSP90.7 regulation with that of other ER chaperones to identify coordinated or divergent responses

• Correlate expression changes with physiological outcomes

Third, normalize HSP90.7 expression data appropriately. For Western blot quantification, normalization to housekeeping proteins like GAPDH is standard , but researchers should verify that normalization controls are not affected by experimental conditions. For immunofluorescence or flow cytometry, appropriate isotype controls and secondary-only controls enable accurate quantification.

Fourth, consider cell type-specific or tissue-specific expression patterns. In plants, HSP90.7 functions in root meristem development , suggesting potential tissue-specific roles. Single-cell analyses or tissue-specific expression studies can provide more nuanced understanding than whole-organism measurements.

What approaches should be used to analyze HSP90.7 knockdown or inhibition experiments?

Analysis of HSP90.7 knockdown or inhibition experiments requires multi-faceted approaches to capture direct and indirect effects:

First, validate knockdown efficiency at both mRNA and protein levels. While siRNA approaches have been successfully used for HSP90 knockdown , protein-level confirmation with HSP90.7 antibodies is essential. Researchers should aim for quantitative assessment, reporting percent reduction compared to controls. In HSP90α/β knockdown studies, even modest knockdown (that didn't affect basal growth) significantly impacted specialized functions like tamoxifen resistance development .

Second, assess both direct molecular consequences and phenotypic outcomes. For HSP90.7, relevant molecular consequences include:

• Changes in client protein stability or localization

• Alterations in ER stress markers

• Effects on interacting pathways (e.g., auxin response proteins in plants )

Phenotypic outcomes might include:

• Growth defects (as observed in HSP90.7 knockout plants )

• Cell viability under stress conditions

• Developmental abnormalities

Third, perform rescue experiments to confirm specificity. Reintroduction of wild-type HSP90.7 should reverse phenotypes caused by specific knockdown, while introduction of mutant versions (e.g., HSP90.7 Δ22 ) may show partial or no rescue. Such experiments provide powerful validation of HSP90.7-specific effects.

Fourth, distinguish between acute and adaptive responses. Short-term HSP90.7 inhibition may reveal immediate chaperone functions, while long-term studies may uncover compensatory mechanisms or more complex physiological roles. Researchers studying HSP90α found that short-term inhibition with ganetespib showed no synergy with tamoxifen in growth assays, but long-term studies revealed profound effects on resistance development .

How can researchers differentiate between direct and indirect effects of HSP90.7 modulation?

Differentiating between direct and indirect effects of HSP90.7 modulation requires experimental designs that establish causality and temporal relationships:

First, implement time-resolved experiments that capture the sequence of events following HSP90.7 modulation. Early events (within minutes to hours) are more likely to represent direct effects, while later events may reflect downstream consequences. Time-course Western blotting, RNA-seq, and functional assays can establish this temporal hierarchy.

Second, identify direct physical interactions using proximity-based approaches. While traditional co-immunoprecipitation with HSP90.7 antibodies can identify stable interactions , transient chaperone-client relationships may require specialized approaches like:

• Crosslinking followed by immunoprecipitation

• Proximity labeling (BioID or APEX) with HSP90.7 as the bait

• FRET-based interaction assays

Third, utilize structure-function analyses with HSP90.7 mutants. Mutations in specific domains can disrupt particular functions while preserving others, helping dissect mechanism. For example, the HSP90.7 Δ22 mutant lacking the plant-specific charged region showed enhanced sensitivity to ER stress despite maintaining general chaperone activity , suggesting domain-specific functions.

Fourth, compare effects of HSP90.7 modulation with those of other ER chaperones or stress inducers. Similar phenotypes may indicate shared pathways, while distinct outcomes suggest HSP90.7-specific functions. This comparative approach can help position HSP90.7 within broader cellular response networks.

Fifth, use pharmacological approaches with different mechanisms. HSP90 inhibitors like 17-AAG have been used to modulate HSP90 activity in cancer research . Comparing genetic knockdown/knockout with chemical inhibition can distinguish between scaffold and enzymatic functions of HSP90.7.

What emerging technologies might enhance HSP90.7 antibody applications in research?

Several emerging technologies hold promise for expanding HSP90.7 antibody applications in future research:

First, single-cell proteomics coupled with HSP90.7 antibodies could reveal cell-to-cell variation in chaperone activity and client interactions. While current Western blot applications provide population averages , single-cell resolution would illuminate how HSP90.7 function varies across cell types and states. Mass cytometry (CyTOF) with metal-conjugated HSP90.7 antibodies could enable multiplexed analysis of HSP90.7 alongside dozens of other proteins at single-cell resolution.

Second, in situ proximity labeling using HSP90.7 antibodies conjugated to enzymes like APEX2 or TurboID would enable comprehensive mapping of the HSP90.7 interactome in intact cells. Unlike traditional co-immunoprecipitation, which may disrupt transient interactions , proximity labeling captures interactions in their native cellular environment and can identify weak or transient clients.

Third, nanobody or single-domain antibody development against HSP90.7 would enable new applications due to their small size and ability to access restricted cellular compartments. These could be particularly valuable for studying ER-localized HSP90.7 in intact cells or for super-resolution microscopy applications.

Fourth, CRISPR-based genetic tagging of endogenous HSP90.7 with split reporters would enable visualization of chaperone-client interactions in real-time. When combined with high-content imaging, this approach could reveal the dynamics of HSP90.7 activity during development or stress responses.

Fifth, cryo-electron microscopy combined with HSP90.7 antibody fragments could reveal structural details of HSP90.7 complexes. This would build on existing knowledge of HSP90 domain structure and provide insights into how the plant-specific charged region affects function.

How might comparative studies across species enhance our understanding of HSP90.7 function?

Comparative studies across species can provide unique insights into HSP90.7 evolution and function:

First, cross-species antibody validation would establish whether currently available HSP90.7 antibodies recognize orthologous proteins in other plant species. This would enable comparative studies of HSP90.7 function across evolutionary distances. Epitope mapping and sequence alignment can guide the selection or development of antibodies with broad cross-reactivity or species-specific recognition.

Second, functional conservation analysis using antibody-based approaches could determine whether the plant-specific charged region in the middle domain of HSP90.7 serves similar functions across plant lineages. Comparing HSP90.7 interactomes between model plants (Arabidopsis) and crop species using identical immunoprecipitation protocols would reveal conserved and species-specific client relationships.

Third, stress response comparisons across species could illuminate how HSP90.7's role in ER stress has evolved. Studies in Arabidopsis have shown that HSP90.7 deletion enhances sensitivity to tunicamycin and calcium stress , but whether this role is conserved in other plants remains unknown. Comparative immunoblotting with HSP90.7 antibodies during stress treatments across species could address this question.

Fourth, developmental phenotype analysis could reveal species-specific adaptations in HSP90.7 function. The role of HSP90.7 in root development and auxin responses in Arabidopsis suggests involvement in fundamental developmental processes, but the extent of functional conservation requires investigation.

A systematic approach comparing HSP90.7 expression, localization, interaction partners, and knockout phenotypes across diverse plant species would provide a comprehensive evolutionary perspective on this essential chaperone.