HSP90-3 Antibody

What are the primary isoforms of HSP90 that antibodies typically target?

HSP90 antibodies generally target one or both of the two principal eukaryotic isoforms:

• HSP90AA1 (HSP90-alpha): This is the inducible form consisting of 732 amino acids (Accession # P07900). Also known as HSP90A, HSPC1, HSPCA, HSP89-alpha, and LAP2 .

• HSP90AB1 (HSP90-beta): This is the constitutively expressed isoform consisting of 724 amino acids (Accession # P08238). Also known as HSP90B, HSPCB, HSPC2, and HSP89-beta .

These isoforms share approximately 90% sequence identity . When selecting an antibody, researchers should verify which isoform(s) the antibody recognizes, as this will impact experimental design and interpretation of results.

What applications are supported by commonly available HSP90 antibodies?

Available HSP90 antibodies support multiple research applications:

• Western Blotting: Typically used at 1:1000 dilution, detecting HSP90 at approximately 90-100 kDa . For example, the HSP90 Antibody #4874 successfully detects endogenous HSP90 protein in various species including human, mouse, and rat samples .

• Immunohistochemistry (Paraffin): Generally used at 1:50 dilution to visualize HSP90 expression in tissue sections . This application is particularly valuable for analyzing HSP90 expression in tumor samples.

• Simple Western: HSP90 can be detected in lysates like HeLa human cervical epithelial carcinoma cell lines using specific antibodies like AF3775 at 5 μg/mL concentration .

When designing experiments, researchers should consider validating antibody performance in their specific sample types and experimental conditions.

How should HSP90 antibodies be stored and handled to maintain optimal activity?

For optimal antibody performance, follow these methodological guidelines:

• Storage temperature: Store at -20°C to -70°C for long-term storage (up to 12 months from receipt) .

• Short-term storage: For reconstituted antibodies, store at 2-8°C under sterile conditions for up to 1 month .

• Long-term storage after reconstitution: Store at -20°C to -70°C under sterile conditions for up to 6 months .

• Avoid freeze-thaw cycles: Use a manual defrost freezer and minimize repeated freeze-thaw cycles which can degrade antibody quality and performance .

Proper handling ensures consistent experimental outcomes and extends the usable lifespan of these research reagents.

What controls should be included when using HSP90 antibodies in experimental protocols?

When designing experiments with HSP90 antibodies, incorporate these methodological controls:

• Positive control: Include cell lines known to express HSP90, such as HeLa cells for Western blotting applications .

• Negative control: When possible, include HSP90-depleted samples or use isotype-matched control antibodies to verify signal specificity.

• Loading control: For Western blotting, include housekeeping proteins (e.g., β-actin, GAPDH) to normalize expression levels.

• Treatment control: When studying HSP90 inhibitors, include untreated samples to establish baseline expression levels for comparison .

These controls help validate experimental results and provide confidence in the specificity and sensitivity of the HSP90 antibody being used.

How can HSP90 antibodies be used to assess the efficacy of HSP90 inhibitors in cancer research?

HSP90 antibodies serve as essential tools for evaluating HSP90 inhibitor efficacy through multiple methodological approaches:

• Client protein degradation: Use anti-HSP90 antibodies alongside antibodies against client proteins (such as Akt, androgen receptor, c-Raf, Cdk4, and Her2) to assess inhibitor-induced client protein destabilization by Western blotting .

• Hsp70 induction measurement: Quantify the compensatory increase in Hsp70 expression (a pharmacodynamic marker) following HSP90 inhibition using anti-Hsp70 antibodies in conjunction with anti-HSP90 antibodies .

• Ex vivo tumor analysis: Apply HSP90 antibodies in immunohistochemistry to assess inhibitor effects in ex vivo cultured human tumor samples, which provides more clinically relevant data than cell line studies alone .

For example, studies with NVP-AUY922 and NVP-HSP990 in prostate cancer showed that a 500 nmol/L treatment resulted in significant client protein degradation (androgen receptor and Akt) and Hsp70 induction, demonstrating on-target HSP90 inhibition .

What methodological approaches can distinguish between high-affinity and low-affinity states of HSP90 in tumor samples?

Distinguishing between HSP90 conformational states requires specialized techniques:

• Competitive binding assays: Extract HSP90 from cell lysates and perform binding assays with labeled inhibitors to quantify the proportion of high-affinity vs. low-affinity HSP90 .

• Co-immunoprecipitation: Use anti-HSP90 antibodies to pull down HSP90 complexes, then probe for associated co-chaperone proteins that indicate the active, high-affinity conformation of HSP90 .

• ATPase activity measurements: Measure HSP90's ATPase activity as a functional readout of its conformational state using biochemical assays with purified HSP90 .

Research has revealed a profound difference (up to 100-fold) in HSP90 binding affinity between normal and malignant cells, which correlates with cell-killing potency of HSP90 inhibitors . This understanding is crucial for developing both therapeutic and diagnostic applications of HSP90 inhibitors.

How can conformation-specific HSP90 antibodies be utilized for cancer diagnostics?

While standard HSP90 antibodies detect total protein levels, conformation-specific antibodies offer enhanced diagnostic potential:

• Identification of activated HSP90: Conformation-specific antibodies could theoretically recognize the high-affinity, activated form of HSP90 found predominantly in cancer cells .

• Diagnostic applications: Such antibodies could identify tumors likely to respond to HSP90 inhibitor therapy, enabling patient stratification and personalized treatment approaches .

• Methodological considerations: These specialized antibodies might recognize epitopes formed by HSP90-co-chaperone interactions or conformation-specific epitopes on HSP90 itself .

While a conformation-specific antibody (9G10) has been reported for Grp94 (the endoplasmic reticulum homolog of HSP90), similar tools specifically for cytosolic HSP90 are still being developed . The development of such antibodies would significantly advance both diagnostic capabilities and our understanding of HSP90 biology in cancer.

What experimental approaches can resolve contradictions between HSP70 induction and biological response to HSP90 inhibitors?

Research has revealed discrepancies between Hsp70 induction (the standard pharmacodynamic marker) and actual biological responses to HSP90 inhibitors:

• Multiplex assessment: Combine HSP70 measurements with client protein degradation analysis (androgen receptor, Akt) using appropriate antibodies to provide a more comprehensive evaluation of inhibitor activity .

• Functional assays: Supplement antibody-based detection with functional readouts such as proliferation inhibition (Ki67 staining) and apoptosis induction (cleaved caspase-3 or TUNEL assay) .

• Ex vivo tissue culture models: Utilize human tumor explants treated with HSP90 inhibitors to assess both target engagement (Hsp70 induction) and biological response in a clinically relevant context .

In prostate cancer studies, 17-AAG, AUY922, and HSP990 all induced Hsp70 equally, but only AUY922 and HSP990 demonstrated significant antiproliferative and proapoptotic effects in ex vivo cultures . This reveals that Hsp70 induction alone is insufficient as a biomarker for predicting therapeutic efficacy.

What are the cross-reactivity profiles of HSP90 antibodies across different species?

Understanding species cross-reactivity is critical for comparative studies and model organism research:

• Multi-species antibodies: Some HSP90 antibodies, like AF3775, recognize HSP90 across human, mouse, and rat species due to high sequence conservation .

• Species-specific antibodies: Other antibodies may be optimized for specific species applications, though the high conservation of HSP90 (up to 100% sequence homology in some regions) often enables cross-species reactivity .

• Validation requirements: When using an antibody in a species not explicitly tested by the manufacturer, researchers should conduct validation experiments to confirm reactivity and specificity .

The HSP90 Antibody #4874, for example, has confirmed reactivity with human, mouse, rat, monkey, D. melanogaster, and zebrafish samples, demonstrating the high conservation of HSP90 across evolutionary diverse species .

How can researchers differentiate between HSP90 isoforms using antibody-based approaches?

Distinguishing between HSP90 isoforms requires careful antibody selection and experimental design:

• Isoform-specific antibodies: Select antibodies raised against unique regions of HSP90AA1 (alpha) or HSP90AB1 (beta) that share minimal sequence homology .

• Western blotting optimization: Due to the similar molecular weights of HSP90 isoforms (HSP90AA1: 732 aa vs. HSP90AB1: 724 aa), use high-resolution gel systems with extended run times to differentiate between the isoforms .

• Co-detection strategies: Use antibodies with different host species or detection systems to simultaneously visualize multiple HSP90 isoforms in the same sample.

When studying HSP90 in functional contexts, researchers should consider that HSP90AA1 is inducible (stress-responsive) while HSP90AB1 is constitutively expressed, which may impact experimental design and interpretation, particularly in stress-response studies .

What methodological considerations are important when using HSP90 antibodies for immunohistochemistry?

For optimal immunohistochemical detection of HSP90 in tissue sections:

• Dilution optimization: Start with a 1:50 dilution as recommended, but optimize for each specific tissue type and fixation method .

• Antigen retrieval: Heat-induced epitope retrieval may be necessary to expose HSP90 epitopes masked by formalin fixation.

• Counterstaining strategies: Counterstain nuclei with DAPI or hematoxylin to assess HSP90 subcellular localization and facilitate quantification.

• Positive controls: Include tissues known to express HSP90 at high levels, such as cancer cell xenografts or certain normal tissues with high HSP90 expression .

In ex vivo cultured prostate cancer tissues, immunohistochemistry with HSP90 and Hsp70 antibodies has successfully demonstrated the differential effects of HSP90 inhibitors on protein expression and localization, validating the utility of this approach in translational research .