HSP90-1 Antibody

What is HSP90 and what are its main functions in cells?

HSP90 is a highly conserved molecular chaperone that assists client proteins in proper folding and stabilization. It represents 1-2% of total mammalian cellular proteins under non-stress conditions and plays essential roles in cellular homeostasis. HSP90 functions through an ATP-dependent cycle that induces conformational changes in client proteins, facilitating their activation .

Key functions include:

• Promoting maturation and structural maintenance of over 200 client proteins

• Participating in cell cycle control and signal transduction pathways

• Supporting cancer cells in overcoming environmental stresses

• Regulating the transcription machinery at multiple levels

• Facilitating malignant transformation in cancer cells

HSP90 engages with client proteins through co-chaperone proteins that act as adapters, simultaneously interacting with both the client protein and HSP90 itself. This forms a functional chaperone complex that completes the protein folding process before releasing the properly folded client protein .

What are the different isoforms of HSP90 and how can antibodies distinguish between them?

In humans, HSP90 comprises five gene isoforms located in different cellular compartments:

HSP90α and HSP90β share approximately 90% sequence identity, making specific antibody generation challenging . To distinguish between isoforms:

• Select antibodies targeting unique epitopes specific to each isoform

• Validate antibody specificity using recombinant proteins and knockout cell lines

• Some antibodies (like AF7247) specifically detect HSP90α with no cross-reactivity with HSP90β

• Other antibodies (like SMC-149) detect both α and β forms equally well

When isoform-specific detection is crucial, verify antibody specificity through western blot analysis using recombinant protein controls for each isoform.

What are the typical dilutions for HSP90-1 antibodies in common applications?

Optimal dilutions for HSP90 antibodies vary by application and specific antibody. Based on commercial antibody documentation, here are recommended ranges:

It is essential to optimize dilutions for each specific antibody, application, and experimental system. Factors influencing optimal dilution include antibody affinity, target abundance, sample type, and detection method. Titration experiments are strongly recommended to determine ideal working concentrations .

How should I validate the specificity of an HSP90-1 antibody?

Comprehensive validation of HSP90 antibody specificity is critical for reliable research results:

• Knockout/knockdown controls: Test the antibody on samples from HSP90 knockout or knockdown cells versus wild-type controls. For example, the AF7247 antibody detected HSP90α in parental HEK293T cells but not in HSP90α knockout HEK293T cells .

• Recombinant protein testing: Evaluate reactivity with recombinant HSP90α and HSP90β to assess isoform specificity. Include dilution series to determine sensitivity thresholds.

• Cross-reactivity assessment: Test against multiple species due to high conservation. Many HSP90 antibodies show cross-reactivity with human, mouse, rat, and other mammalian species .

• Multiple detection methods: Validate using different techniques (WB, IHC, IF) to ensure consistent results across applications.

• Molecular weight verification: Confirm detection at the expected molecular weight (85-90 kDa) and assess for non-specific bands.

• Peptide competition: Pre-incubate antibody with immunizing peptide/protein to demonstrate binding specificity through signal blocking.

Proper validation increases confidence in antibody specificity and experimental reproducibility, particularly important for distinguishing between closely related HSP90 isoforms.

How can I discriminate between activated (high-affinity) and inactive (low-affinity) conformations of HSP90 using antibodies?

HSP90 exists in different conformational states that correlate with its functional activity. The high-affinity conformation prevalent in cancer cells versus the low-affinity form in normal cells presents both challenges and opportunities for research :

Methodological approaches:

• Conformation-specific antibodies:

  • A conformation-specific antibody (9G10) exists for Grp94 (ER homolog of HSP90)

  • Development of similar antibodies for cytosolic HSP90 would be valuable

  • Such antibodies might recognize epitopes formed by HSP90-co-chaperone interactions

• Co-chaperone association analysis:

  • The activated HSP90 conformation involves complex formation with co-chaperones

  • Co-immunoprecipitation of HSP90 followed by blotting for co-chaperones (p23, Cdc37, etc.)

  • Higher levels of co-precipitating proteins indicate the active, complexed form

• ATP/ADP-dependent experimental designs:

  • HSP90 conformational changes are ATP/ADP-dependent

  • Use ATP/ADP analogs to lock HSP90 in specific conformations before antibody detection

  • Compare antibody binding patterns under different nucleotide conditions

• Competitive binding assays:

  • Differential binding affinities between normal and malignant cells can be detected using competitive binding assays with HSP90 inhibitors

  • Combine with antibody-based detection methods for characterizing activation state

These approaches can help distinguish between the different conformational states of HSP90, which has significant implications for cancer biology and drug development.

What are the best experimental approaches to study extracellular HSP90 (eHSP90) using antibodies?

Extracellular HSP90 (eHSP90) plays crucial roles in tumor metastasis and angiogenesis. Here are methodological approaches to study eHSP90 using antibodies:

• Detection and quantification of eHSP90:

  • ELISA: Use capture antibodies (like scFv47) to detect eHSP90 in culture media or biological fluids

  • Western blotting: Analyze concentrated cell culture supernatants, confirming no cellular contamination by probing for cytoplasmic markers like tubulin

  • Surface Plasmon Resonance (SPR): For quantitative binding analysis of eHSP90 in real-time

• Functional inhibition studies:

  • Membrane-impermeable antibodies can specifically block eHSP90 without affecting intracellular HSP90

  • Compare effects of eHSP90 blocking versus total HSP90 inhibition on cancer cell functions

  • Single-chain antibody fragments (scFvs) can effectively recognize extracellular Hsp90 in breast cancer models

• Cell surface localization:

  • Immunofluorescence on non-permeabilized cells detects surface-associated eHSP90

  • Flow cytometry on non-permeabilized cells using anti-HSP90 antibodies quantifies surface expression

  • Live cell imaging with fluorescently labeled antibodies tracks dynamic changes

Experimental design considerations:

• Include controls to distinguish secreted HSP90 from leaked intracellular HSP90

• Compare cancer cells with non-cancer cells (studies show lower eHSP90 in non-cancer fibroblast BJ cells)

• Use serum-free conditions when collecting secreted proteins to avoid interference

• Note that eHSP90α is a C-terminal truncated form compared to intracellular HSP90α

These approaches enable comprehensive investigation of eHSP90 biology and its potential as a therapeutic target in cancer metastasis.

How can HSP90-1 antibodies be used to investigate HSP90's role in cancer progression?

HSP90 plays critical roles in cancer by stabilizing numerous oncogenic client proteins. Here are methodological approaches using HSP90 antibodies to investigate these roles:

• Expression and localization analysis:

  • Immunohistochemistry (IHC) of tumor tissue microarrays to correlate HSP90 expression with clinical outcomes

  • Immunofluorescence to determine subcellular localization changes during cancer progression

  • Western blotting to quantify expression levels across cancer stages or after treatment

  • Flow cytometry to analyze HSP90 levels in circulating tumor cells

• Client protein relationship studies:

  • Co-immunoprecipitation to identify cancer-specific HSP90 client proteins

  • Proximity ligation assays to visualize HSP90-client interactions in situ

  • Monitor client protein stability after HSP90 inhibition via western blotting

  • Follow the "HSP90 addiction" phenomenon in cancer cells by correlating HSP90 activity with client protein function

• Extracellular HSP90 in metastasis:

  • Quantify secreted HSP90 in patient samples via ELISA

  • Functional studies using antibody blocking of eHSP90 in invasion/migration assays

  • Track eHSP90-client interactions in the tumor microenvironment

• HSP90 conformation in tumors:

  • Investigate the high-affinity, activated HSP90 conformation predominant in tumors versus normal cells

  • Correlate HSP90 activation state with therapeutic responses

  • Examine how stress conditions in the tumor microenvironment affect HSP90 conformational states

The search results highlight that cancer cells demonstrate a significantly higher proportion of HSP90 in an activated, high-affinity conformation compared to normal cells (up to 100-fold difference) . This presents a valuable therapeutic window for targeting HSP90 in cancer treatment while potentially minimizing effects on normal tissues.

What methodological approaches can resolve contradictory data when using different HSP90 antibodies?

Contradictory results with different HSP90 antibodies can arise from various factors. Here are methodological approaches to resolve such discrepancies:

• Comprehensive antibody characterization:

  • Epitope mapping to determine precise binding regions for each antibody

  • Testing on recombinant HSP90 isoforms to confirm specificity profiles

  • Validation in knockout/knockdown models to ensure specificity

  • Cross-reactivity assessment against related heat shock proteins

• Comparative analysis framework:

  • Use multiple antibodies targeting different HSP90 epitopes in parallel experiments

  • Include both monoclonal and polyclonal antibodies when possible

  • Document lot-to-lot variation by maintaining reference samples

  • Compare results across different applications (WB, IHC, IP)

• Technical resolution strategies:

  Issue	Resolution Approach	Validation Method
  Isoform-specific reactivity	Use recombinant HSP90α and HSP90β controls	Western blot with recombinant proteins
  Conformation-dependent epitopes	Test both native and denatured samples	Parallel native and SDS-PAGE
  Post-translational modification masking	Include phosphatase/deglycosylation treatments	Compare treated vs. untreated samples
  Cross-reactivity issues	Perform peptide competition assays	Pre-absorption with immunizing peptide
  Clone-specific artifacts	Use alternative antibody clones	Multiple antibody approach

• Orthogonal verification:

  • Confirm findings with non-antibody methods where possible

  • Use mass spectrometry for protein identification in complex samples

  • Apply genetic approaches (knockdown/knockout) to verify antibody specificity

  • Consider expressing tagged HSP90 for independent detection

When publishing research, document all antibody information (supplier, catalog number, lot, dilution, validation method) to facilitate reproducibility and proper interpretation of results.

How can I optimize immunoprecipitation protocols with HSP90-1 antibodies for studying novel client proteins?

Optimizing immunoprecipitation (IP) protocols to identify novel HSP90 client proteins requires careful consideration of factors that maintain complex integrity:

• Antibody selection considerations:

  • Choose antibodies validated specifically for IP applications (documented in product data sheets)

  • Consider epitope location to avoid interfering with client binding regions

  • Compare multiple antibodies targeting different epitopes to confirm results

  • Balance between monoclonal specificity and polyclonal coverage

• Lysis buffer optimization:

  • Use gentle non-ionic detergents (0.5-1% NP-40 or Triton X-100)

  • Test different salt concentrations (typically 100-150 mM NaCl)

  • Include protease and phosphatase inhibitors to preserve interactions

  • Consider ATP concentration (1-5 mM) to stabilize certain interactions

  • Test with/without sodium molybdate (20 mM) which stabilizes HSP90 complexes

• IP procedure refinement:

  • Pre-clear lysates to reduce non-specific binding

  • Optimize antibody amounts (0.5-4.0 μg per 1-3 mg lysate)

  • Compare different incubation conditions (4°C overnight vs. 1-2 hours)

  • Evaluate crosslinking strategies for capturing transient interactions

  • Optimize wash stringency based on complex stability

• Client validation approaches:

  • Treat cells with HSP90 inhibitors before IP to confirm true client relationships

  • Compare normal versus heat-shocked cells to observe stress-dependent interactions

  • Test ATP dependence using ATP versus non-hydrolyzable analogs

  • Perform reciprocal IP using antibodies against suspected client proteins

  • Verify direct interaction using in vitro reconstitution with purified components

These methodological considerations enable robust identification and validation of novel HSP90 client proteins, expanding our understanding of HSP90's diverse cellular functions.

How can I design experiments using HSP90-1 antibodies to investigate its role in viral infections?

HSP90 plays critical roles in viral replication, with viruses utilizing host HSP90 to facilitate their assembly and life cycle. Here's how to design experiments to investigate this role:

• Viral protein-HSP90 interaction studies:

  • Co-immunoprecipitation using HSP90 antibodies to pull down viral proteins

    • Example targets: HBV reverse transcriptase, HCV NS2-3 protease, HSV-1 proteins

  • Proximity ligation assays to visualize interactions in infected cells

  • Test effects of HSP90 inhibitors on these interactions (geldanamycin shows broad antiviral activity)

• Functional impact assessment:

  • Compare selective knockdown of HSP90 isoforms on viral replication

  • Monitor viral protein stability and function with/without HSP90 inhibition

  • Quantify viral titers under conditions of HSP90 modulation

  • Test HSP90 antibody blocking effects on viral entry or assembly

• Localization studies:

  • Immunofluorescence co-localization of HSP90 with viral components during infection

  • Track changes in HSP90 distribution during the viral life cycle

  • Analyze whether HSP90 relocates to viral replication sites

• HSP90 conformation during infection:

  • Assess whether viral infection induces high-affinity HSP90 conformations similar to cancer

  • Compare HSP90-co-chaperone complexes in infected versus uninfected cells

  • Determine if viral proteins induce specific conformational changes in HSP90

Research suggests significant therapeutic potential for HSP90 inhibitors against viruses. For example, geldanamycin inhibited HSV-1 replication with an IC50 of 93 nM, while requiring much higher concentrations (IC50 of 350 μM) to affect uninfected cells, suggesting excellent selectivity . This differential sensitivity parallels the cancer-normal cell distinction in HSP90 utilization.

What are the technical challenges in using HSP90-1 antibodies for in vivo imaging or targeted therapy research?

Using HSP90 antibodies for in vivo imaging or targeted therapy presents several technical challenges requiring methodological solutions:

• Antibody format optimization:

  • Full antibodies (150 kDa) have limited tissue penetration

  • Single-chain antibody fragments (scFvs, ~25 kDa) offer better tissue distribution

  • The selection and characterization of scFvs specific to HSP90α from phage display libraries has been demonstrated and validated

  • These smaller formats maintain specificity while improving pharmacokinetic properties

• Target specificity challenges:

  • Distinguishing intracellular vs. extracellular HSP90

    • Design membrane-impermeable antibodies targeting extracellular HSP90 (eHSP90)

    • Focus on the secreted form of HSP90 present in breast cancer models

  • Isoform selectivity between HSP90α and HSP90β

    • Carefully selected epitopes can provide isoform specificity

    • Measure binding affinities (KD) to ensure specificity (one study found a scFv with 1.5-fold higher affinity for HSP90β than HSP90α)

  • Normal vs. tumor HSP90 selectivity

    • Target the high-affinity, activated conformation predominantly found in tumors

• Therapeutic application considerations:

  • Extracellular targeting strategy

    • Antibodies as cell-impermeable inhibitors specifically blocking eHSP90 functions

    • Potential for treating cancers where invasiveness and metastasis depend on eHSP90 secretion

  • Combination approaches

    • Design studies examining synergistic effects with conventional HSP90 inhibitors

    • Target different pools of HSP90 simultaneously (intracellular and extracellular)

• Validation requirements:

  • Demonstrate specific binding to HSP90 in vivo through ex vivo analysis

  • Confirm correlation between imaging signal and HSP90 levels

  • Establish relationship between antibody binding and functional outcomes

  • Thorough toxicity evaluation in multiple tissues

The search results highlight that antibody-based inhibitors targeting extracellular HSP90 could represent "a new class of cell-impermeable inhibitors" with potential therapeutic applications in multiple cancer types including breast cancer, melanoma, fibrosarcoma, colorectal cancer, and glioblastoma .