HSP90-4 Antibody

What are the primary applications for HSP90 antibodies in research?

HSP90 antibodies are versatile tools with multiple research applications, including western blotting (WB), immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). For instance, the HSP90 Antibody (4F10) is specifically documented for detecting human HSP90 proteins through these methods . Additionally, certain HSP90 antibodies are suitable for immunohistochemistry on paraffin-embedded samples (IHC-P), allowing researchers to examine HSP90 expression patterns in tissue sections .

The wide species cross-reactivity of many commercially available HSP90 antibodies (human, mouse, rat, monkey, D. melanogaster, zebrafish) makes them valuable for comparative studies across model organisms . When selecting an HSP90 antibody for your research, consider the specific experimental application, target species, and antibody format (monoclonal vs. polyclonal) to ensure optimal results.

How should researchers validate the specificity of HSP90 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For HSP90 antibodies, consider implementing these methodological approaches:

• Western blot analysis with positive and negative controls, looking for the expected 90 kDa band

• Peptide competition assays to confirm binding specificity

• Knockdown/knockout validation using siRNA or CRISPR methods

• Cross-validation with multiple antibodies targeting different HSP90 epitopes

When evaluating an HSP90 antibody, it's important to note that certain antibodies, such as AC88, specifically recognize uncomplexed HSP90 . This property can be leveraged for examining the proportion of free versus complexed HSP90 in experimental samples, but could also represent a limitation if total HSP90 detection is desired.

What are the recommended sample preparation methods for HSP90 antibody-based experiments?

Proper sample preparation is essential for successful HSP90 antibody experiments. For cell lysate preparation:

• Use buffers containing protease inhibitors to prevent degradation

• Include phosphatase inhibitors when studying HSP90 phosphorylation status

• Consider gentle lysis methods that preserve protein-protein interactions if studying HSP90 complexes

• Maintain sample temperature below 4°C throughout processing to preserve HSP90 conformational state

For immunohistochemistry applications, optimal fixation and antigen retrieval protocols are critical. For HSP90 antibodies like the one from Cell Signaling Technology, a dilution of 1:50 is recommended for IHC-P applications . When preparing samples for mass spectrometry analysis of HSP90-associated proteins, consider approaches like isotopic labeling (ICAT or iTRAQ) to enable comparative analyses between different treatment conditions .

How can researchers distinguish between different HSP90 conformational states using antibodies?

HSP90 exists in different conformational states that correlate with its functional cycle and ATP binding status. Distinguishing between these states is critical for studying HSP90's chaperone activity.

Research has revealed that tumors contain HSP90 in a high-affinity conformation that differs from the predominantly low-affinity conformation found in normal cells . While conformation-specific antibodies for HSP90 have been challenging to develop, the identification of such tools represents an important frontier in HSP90 research.

The following methodological approaches can help researchers investigate HSP90 conformational states:

• Competitive binding assays using known HSP90 inhibitors

• Co-immunoprecipitation with conformation-dependent co-chaperone proteins

• ATPase activity assays coupled with antibody recognition patterns

• Cross-linking approaches to stabilize specific conformational states prior to antibody application

The development of conformation-specific antibodies, similar to the 9G10 antibody for the HSP90 homolog Grp94, would significantly advance the field's ability to study HSP90 conformational dynamics in different biological contexts .

What are the optimal approaches for studying HSP90-client protein interactions?

Understanding HSP90-client protein interactions is fundamental to elucidating HSP90's cellular functions. Several methodological approaches can be employed:

Advanced mass spectrometry approaches, including quantitative spectrum counting and isotope labeling techniques like ICAT and iTRAQ, have been successfully applied to identify statistically significant changes in the HSP90-bound proteome under different conditions . These techniques allow for the simultaneous identification of hundreds to thousands of HSP90-associated proteins.

For validation of potential HSP90 client proteins identified in high-throughput screens, researchers should consider multiple criteria including: re-identification of known co-chaperone partners, identification of multiple components of individual cellular pathways, overlaps between discrete assays performed in separate labs, and detailed characterization of individual novel interactions .

How can researchers investigate HSP90's role in immune responses for cancer immunotherapy development?

HSP90's role in immune responses presents opportunities for cancer immunotherapy development. Recent research has identified HSP90-derived MHC class II epitopes as potential cancer vaccine candidates.

A study demonstrated that specific HSP90 peptides (p485 and p527) could induce strong antigen-specific T cell responses through cross-priming of CD8+ T cells in vivo . These peptides showed promising results in established tumor models, particularly when combined with STING agonists and/or anti-CTLA-4 antibodies.

Methodological approaches for investigating HSP90's immunological potential include:

• In silico algorithms to predict MHC class II epitopes with high binding affinities

• ELISPOT assays to validate epitope-specific T cell responses

• In vivo tumor models to evaluate antitumor efficacy

• Multiplex immunohistochemistry to assess the immune microenvironment

• TCRβ sequencing to examine T cell repertoire diversity

The increased tumor rejection observed with HSP90 peptide vaccines was associated with enhanced systemic HSP90-specific T-cell responses, increased T-cell recruitment to the tumor microenvironment, intermolecular epitope spreading, and increased TCRβ rearrangement .

What strategies can address non-specific binding of HSP90 antibodies?

Non-specific binding is a common challenge when working with HSP90 antibodies. To minimize this issue:

• Optimize blocking conditions by testing different blocking agents (BSA, non-fat milk, commercial blockers)

• Increase the stringency of washing steps using buffers with appropriate detergent concentrations

• Titrate primary antibody concentrations to find the optimal signal-to-noise ratio

• Consider using monoclonal antibodies like HSP90 Antibody (4F10) that offer high specificity

• Include appropriate negative controls in each experiment

When performing western blotting, recommended dilutions for HSP90 antibodies typically range from 1:1000 to 1:5000, depending on the specific antibody and application. For immunohistochemistry, more concentrated antibody solutions (approximately 1:50) are generally required .

How can researchers effectively measure HSP90 inhibition in response to therapeutic agents?

Measuring HSP90 inhibition is crucial for evaluating potential therapeutic agents. Several methodological approaches can be employed:

• Client protein degradation assays: Monitor levels of known HSP90 client proteins (e.g., HER2, Akt, Raf-1) following treatment with inhibitors

• HSP90 binding assays: Use competitive binding assays to measure inhibitor affinity for HSP90

• Cellular thermal shift assays (CETSA): Evaluate thermal stabilization of HSP90 upon inhibitor binding

• ATPase activity assays: Measure inhibition of HSP90's intrinsic ATPase activity

• Co-chaperone association: Assess changes in HSP90-cochaperone interactions

Research has demonstrated that Hsp90 inhibitors like 17-allylaminogeldanamycin (17-AAG) and macbecin II can cause the dissociation of HSF-1 complexes and trigger a robust heat-shock response . This response may create a time window for cytotoxic activity that could be limited by the development of drug-induced heat-shock responses. Understanding these dynamics is essential for designing effective therapeutic strategies targeting HSP90.

What are the best practices for quantitative analysis of HSP90 expression using antibody-based methods?

Quantitative analysis of HSP90 expression requires careful methodological considerations:

• Include appropriate loading controls for normalization

• Establish a standard curve using recombinant HSP90 for absolute quantification

• Use digital image analysis software with appropriate background correction

• Consider multiplexed approaches to simultaneously detect HSP90 and relevant markers

• Validate findings with orthogonal techniques (e.g., qPCR, mass spectrometry)

When analyzing HSP90 in tumor samples versus normal tissues, it's important to note that while total HSP90 levels may not differ dramatically, the conformational state and complexes formed can vary significantly . This functional difference is reflected in the 100-fold difference in HSP90 binding affinity observed between normal and malignant cells, which correlates with cell-killing potency of HSP90 inhibitors .

How can HSP90 antibodies be utilized in developing novel cancer diagnostics?

The unique properties of HSP90 in cancer cells present opportunities for diagnostic applications. Research has identified profound differences in HSP90 activity between normal and malignant cells, with a 100-fold difference in binding affinity and percentage of free versus complexed HSP90 .

Methodological approaches for developing HSP90-based cancer diagnostics include:

• Developing conformation-specific antibodies that selectively recognize the high-affinity form of HSP90 found in tumors

• Creating immunoassays that can determine HSP90 usage patterns in clinical samples

• Combining HSP90 detection with other cancer biomarkers for improved diagnostic accuracy

• Establishing standardized protocols for sample preparation and analysis

While the AC88 antibody recognizes uncomplexed HSP90 and might be useful for measuring decreased HSP90 usage, an ideal reagent would be an antibody specifically recognizing the high-affinity conformation of HSP90 found in tumors . Such an antibody might detect an epitope formed by the close association of HSP90 with co-chaperone proteins or recognize an activated conformation-specific epitope on HSP90 itself.

What role do post-translational modifications of HSP90 play in antibody recognition and function?

HSP90 undergoes various post-translational modifications (PTMs) that affect its chaperone function, client protein interactions, and conformational states. These modifications can impact antibody recognition and functional studies:

For researchers studying HSP90 PTMs, phosphospecific or modification-specific antibodies can provide valuable insights into the regulatory mechanisms controlling HSP90 function. When interpreting results from these studies, it's important to consider how sample preparation methods might preserve or alter these modifications.

How can researchers leverage HSP90 antibodies for developing peptide-based cancer vaccines?

HSP90-targeted immunotherapeutic approaches represent an emerging area of cancer research. The development of peptide-based vaccines targeting HSP90 has shown promising results in preclinical models .

Methodological approaches for this research direction include:

• Using in silico algorithms to identify HSP90-derived MHC class II epitopes with high binding affinities across multiple human HLA class II genotypes

• Validating candidate epitopes using ELISPOT assays to assess T-cell responses

• Evaluating antitumor efficacy in appropriate animal models

• Combining HSP90 peptide vaccines with immune adjuvants or checkpoint inhibitors to enhance efficacy

Research has identified HSP90-derived peptides (p485 and p527) as promising Th1 immunity-inducing epitopes that can trigger strong antigen-specific T cell responses through cross-priming of CD8+ T cells in vivo . The efficacy of these peptide vaccines was enhanced when combined with STING agonists and/or anti-CTLA-4 antibodies, leading to increased tumor rejection associated with enhanced systemic HSP90-specific T-cell responses .