HSP90B1 P94B9AT Antibody

What is HSP90B1 and why is it significant in cancer research?

HSP90B1, also known as Grp94, is a molecular chaperone belonging to the heat shock protein 90 family. It plays a crucial role in cellular processes by facilitating the degradation and removal of misfolded proteins and impaired organelles through autophagy mechanisms. Its significance in cancer research stems from its overexpression in various cancers, including head and neck squamous cell carcinoma (HNSC), where it correlates with advanced disease stages and poor prognosis . HSP90B1 interacts with over 100 different client proteins and contributes to tumor adaptation to microenvironmental stressors such as glucose deficiency, hypoxia, acidosis, and immune stimulation .

What are the optimal applications for HSP90B1 antibodies in experimental research?

HSP90B1 antibodies are versatile tools applicable across multiple experimental techniques. Based on verified samples and recommended protocols, HSP90B1 antibodies demonstrate high utility in Western blot (WB) analyses at dilutions of 1:500-1:2000, immunohistochemistry (IHC) at 1:50-1:100, and immunofluorescence (IF) at 1:50-1:100 . These antibodies have been successfully validated in various cell lines (A375, HeLa, L-O2, NIH/3T3) and tissue samples (mouse liver, rat testis, human lung cancer, mouse kidney), confirming their reliability across human, mouse, and rat samples . For protein expression quantification, Western blot represents the gold standard, while IHC provides valuable insights into tissue localization patterns.

How should researchers validate HSP90B1 antibody specificity for their experimental models?

Methodological approach to antibody validation should include:

• Positive and negative controls: Use cell lines with known HSP90B1 expression levels as verified in previous studies, such as TU686 and Fadu (high expression) compared to normal human oral keratinocytes (HOK, lower expression) .

• Knockdown verification: Generate HSP90B1 knockdown models (using siRNA or shRNA approaches as described in research) to confirm antibody specificity by demonstrating reduced signal in Western blot or IHC .

• Cross-reactivity testing: When working with non-human models, confirm reactivity across species using appropriate positive controls in mouse and rat samples .

• Multiple detection methods: Validate expression using complementary techniques such as qRT-PCR to correlate protein detection with mRNA expression levels .

What are the recommended storage and handling conditions for HSP90B1 antibodies?

For optimal performance and longevity of HSP90B1 antibodies, researchers should adhere to the following methodological practices:

• Store antibodies at -20°C for long-term storage, avoiding repeated freeze-thaw cycles

• For working solutions, store at 4°C for up to one month

• Prepare aliquots of concentrated antibody to minimize freeze-thaw cycles

• Always centrifuge briefly before opening vials to collect solution at the bottom

• For dilution, use appropriate buffers as recommended in specific protocols (typically PBS with 0.1% BSA)

• When performing Western blot, IHC, or IF, optimize blocking conditions to minimize background signal

How can HSP90B1 antibodies be utilized to investigate autophagy mechanisms in cancer models?

Investigating HSP90B1's role in autophagy requires sophisticated methodological approaches:

• Dual immunofluorescence staining: Co-localize HSP90B1 with autophagy markers such as LC3B to visualize interaction points. Use HSP90B1 antibodies at 1:50-1:100 dilution for immunofluorescence .

• Autophagy flux assessment: Combine HSP90B1 detection with autophagy inhibitors (chloroquine or bafilomycin A1) to distinguish between autophagy induction and blockade.

• Protein interaction studies: Perform co-immunoprecipitation with HSP90B1 antibodies to identify binding partners within the PI3K/AKT/mTOR signaling pathway .

• Functional validation: Use HSP90B1 knockdown and overexpression models to measure autophagy markers (LC3-I/LC3-II conversion, p62/SQSTM1 levels) by Western blot to establish causality .

Research has demonstrated that HSP90B1 obstructs autophagy and promotes HNSC progression through the PI3K/Akt/mTOR pathway, suggesting that targeting this interaction could present therapeutic opportunities .

What are the key considerations when designing experiments to investigate HSP90B1's role in cancer cell proliferation and metastasis?

When designing experiments to evaluate HSP90B1's influence on cancer progression, researchers should consider:

What strategies should be employed to address potential data inconsistencies when quantifying HSP90B1 expression across different experimental platforms?

Resolving data inconsistencies requires methodological rigor:

• Standardization of detection methods:

  • For Western blot: Implement normalization to multiple housekeeping proteins

  • For IHC: Use standardized scoring systems that account for both staining intensity and proportion of positive cells

  • For qRT-PCR: Employ multiple reference genes for normalization

• Cross-platform validation: Confirm expression patterns using complementary techniques:

  • Correlate protein levels (WB/IHC) with mRNA expression (qRT-PCR)

  • Compare in vitro findings with patient tissue analyses

• Statistical approaches: Apply appropriate statistical methods to handle variability:

  • For smaller sample sizes: Non-parametric tests

  • For larger datasets: Consider data transformation when assumptions of normality are violated

• Reference databases: Validate findings against public databases:

  • Use GEPIA database (http://gepia.cancer-pku.cn/) to compare expression in cancerous versus normal tissues

  • Correlate with survival data from Kaplan–Meier plotter database

How can HSP90B1 antibodies be leveraged in conjunction with other markers to develop comprehensive prognostic panels for HNSC patients?

Developing prognostic panels requires sophisticated methodological integration:

• Multiplex immunohistochemistry:

  • Combine HSP90B1 (1:50-1:100 dilution) with additional markers including:

    • Autophagy markers (LC3B, p62)

    • PI3K/AKT/mTOR pathway components

    • Proliferation markers (Ki-67)

    • Apoptosis markers (Cleaved caspase-3)

• Multivariate analysis frameworks:

  • Integrate HSP90B1 expression with clinicopathological factors shown to correlate with outcomes:

    • T-stage and M-stage (significant correlation with HSP90B1 expression, p=0.037 and p=0.001, respectively)

    • Clinical stage (p=0.033)

    • Differentiation grade

• Prognostic algorithm development:

  • Utilize the prognostic correlation already established (patients with elevated HSP90B1 levels experienced significantly reduced survival times)

  • Develop weighted scoring systems incorporating both molecular markers and clinical parameters

• Validation cohorts:

  • Test prognostic panels across diverse patient populations

  • Validate using both retrospective and prospective approaches

What are the most common technical challenges when working with HSP90B1 antibodies in immunohistochemistry, and how can they be addressed?

Common technical challenges in IHC with HSP90B1 antibodies include:

• Background staining issues:

  • Methodological solution: Optimize blocking conditions using 3% hydrogen peroxide (one hour) to block endogenous peroxidase activity

  • Increase blocking duration or concentration of blocking agent

  • Use species-specific blocking reagents

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval (HIER) and enzymatic retrieval methods

  • Adjust pH conditions (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Optimize retrieval duration based on tissue fixation conditions

• Antibody concentration optimization:

  • Perform titration experiments using the recommended 1:50-1:100 dilution range

  • Include positive control tissues (verified samples: rat testis, human lung cancer, mouse kidney)

• Signal amplification for low-expressing samples:

  • Implement polymer-based detection systems

  • Consider tyramide signal amplification for challenging samples

  • Optimize visualization using DAB reagent with standardized development times

How should researchers interpret and analyze HSP90B1 expression data in relation to clinicopathological features?

Methodological approach to data interpretation should include:

• Standardized scoring systems:

  • Implement dual-parameter scoring considering both staining intensity and proportion of positive cells

  • Use independent evaluations by multiple pathologists to minimize subjective bias

• Statistical analysis framework:

  • Apply chi-square tests for categorical variables (as demonstrated in Table 2 of the research)

  • Use appropriate parametric or non-parametric tests for continuous variables

  • Consider multivariate analysis to adjust for confounding factors

• Correlation with clinical parameters:

  • Systematically analyze association with:

    • Survival status (demonstrated significance p=0.031)

    • T stage (p=0.037)

    • M stage (p=0.001)

    • Tumor grade (p=0.004)

    • Clinical stage (p=0.033)

• Presentation of results:

  • Present data in standardized tables similar to Tables 1 and 2 in the referenced research

  • Include appropriate statistical measures (p-values, confidence intervals)

  • Consider Kaplan-Meier survival curves for prognostic correlations

What quality control measures should be implemented when using HSP90B1 antibodies across different experimental batches?

Rigorous quality control requires:

• Reference standard inclusion:

  • Maintain consistent positive controls across experiments

  • Include cell lines with known expression levels (TU686, Fadu, SAS, HOK)

• Batch normalization strategies:

  • Include internal reference samples in each experimental batch

  • Apply appropriate normalization factors to adjust for batch effects

  • Consider replicate testing across batches for high-priority samples

• Antibody validation for each new lot:

  • Perform Western blot validation with known positive samples

  • Compare staining patterns across antibody lots

  • Document lot-specific optimal working dilutions

• Standardized protocols:

  • Maintain strict adherence to established protocols

  • Document any deviations or modifications

  • Use automated systems where possible to reduce technical variability

How might combining HSP90B1 antibodies with other molecular tools advance understanding of autophagy regulation in cancer?

Future research methodologies should explore:

• Proximity ligation assays (PLA):

  • Combine HSP90B1 antibodies with antibodies against key interacting proteins

  • Visualize direct protein-protein interactions in situ

  • Map interaction networks in different cellular compartments

• CRISPR-based functional genomics:

  • Generate HSP90B1 domain-specific mutations to identify functional regions

  • Create cellular models with modified HSP90B1 phosphorylation sites

  • Correlate structural modifications with changes in autophagy markers

• Single-cell analysis platforms:

  • Apply HSP90B1 antibodies in single-cell protein profiling

  • Identify cell subpopulations with distinct HSP90B1 expression patterns

  • Correlate with single-cell transcriptomics data

• Live-cell imaging technologies:

  • Develop fluorescently tagged HSP90B1 constructs

  • Monitor real-time dynamics of HSP90B1 during autophagy induction

  • Correlate with autophagosome formation and clearance

What are the emerging applications of HSP90B1 antibodies in developing targeted therapeutic approaches for HNSC?

Therapeutic development methodologies include:

• Companion diagnostic development:

  • Standardize HSP90B1 IHC protocols for patient stratification

  • Establish clinically relevant expression thresholds

  • Correlate expression patterns with response to specific therapies

• Drug screening platforms:

  • Use HSP90B1 antibodies to monitor target engagement of novel inhibitors

  • Develop cell-based assays for high-throughput screening

  • Validate hits using functional assays established in previous research

• Combination therapy rational design:

  • Target HSP90B1 in conjunction with PI3K/AKT/mTOR pathway inhibitors

  • Monitor pathway modulation using phospho-specific antibodies

  • Assess synergistic effects on autophagy and apoptosis markers

• Antibody-drug conjugate development:

  • Explore HSP90B1 as a potential target for antibody-drug conjugates

  • Assess internalization dynamics

  • Optimize linker chemistry and payload selection