HSP90AB1 Recombinant Monoclonal Antibody

What is HSP90AB1 and what biological significance does it have in research?

HSP90AB1, also known as HSP90 beta, is a molecular chaperone protein belonging to the HSP90 family, which includes cytosolic HSP90 alpha (HSP90AA1), endoplasmic reticulum-related GRP74, and mitochondrial TRAP1 . It plays crucial roles in cellular processes including protein folding, maturation, activation, and degradation . HSP90AB1 is particularly significant in research because it participates in the regulation of multiple signaling pathways involved in cell cycle progression, cell survival, and apoptosis .

The protein interacts with a wide range of client proteins, including many oncogenic proteins, making it an important target for cancer therapy research . Additionally, HSP90AB1 is involved in cellular responses to environmental stressors such as heat shock and oxidative stress, with its expression being induced by various stress stimuli . Upregulation of HSP90AB1 has been observed across various cancer types, making it a valuable biomarker in oncology research .

What applications are HSP90AB1 recombinant monoclonal antibodies suitable for?

HSP90AB1 recombinant monoclonal antibodies are validated for multiple research applications, with performance characteristics varying by manufacturer and clone. Based on current validation data, these antibodies are suitable for:

For optimal results, researchers should note that different antibody clones demonstrate varying reactivity profiles. For example, while some antibodies like Cusabio's CSB-RA904894A0HU specifically detect only human HSP90AB1 , others such as Proteintech's 80301-1-RR show cross-reactivity with mouse, rat, and zebrafish samples , and OriGene's OTI4C10 clone reacts with dog, human, monkey, mouse, and rat specimens .

How should I determine the optimal dilution for my specific experimental system?

Determining the optimal dilution for HSP90AB1 antibodies requires systematic titration within your experimental system, as recommended dilutions provided by manufacturers represent starting points rather than definitive values . For a methodical approach:

• Begin with a dilution series spanning the manufacturer's recommended range (e.g., for Western blot, test 1:1000, 1:5000, 1:10000, and 1:20000 if the recommended range is 1:5000-1:50000)

• Consider sample-specific factors:

  • Cell/tissue type (different cell lines show variable HSP90AB1 expression levels)

  • Protein extraction method (affects protein availability and epitope integrity)

  • Detection system sensitivity (chemiluminescent vs. fluorescent)

• Perform validation experiments with positive controls from validated cell lines:

  • For Western blot: HeLa, HEK-293, HepG2, Jurkat, NIH/3T3, and Neuro-2a cells have been confirmed as positive controls

  • For IHC: Human breast cancer tissue has been validated with appropriate antigen retrieval methods

  • For IF/ICC: HeLa cells demonstrate reliable signal

• Evaluate signal-to-noise ratio at each dilution, selecting the concentration that provides maximum specific signal with minimal background

Remember that antibody performance can vary between lots and may require re-optimization when changing experimental conditions or when using fresh reagent batches .

What factors should I consider when selecting between different HSP90AB1 antibody clones?

When selecting an HSP90AB1 antibody clone for research, consider these critical factors to ensure optimal experimental outcomes:

• Target species reactivity:

  • CSB-RA904894A0HU detects only human HSP90AB1

  • 80301-1-RR shows reactivity with human, mouse, rat, and zebrafish samples

  • OTI4C10 demonstrates broader reactivity with dog, human, monkey, mouse, and rat specimens

  • 67450-1-Ig targets human, mouse, and rat HSP90AB1

• Immunogen design and epitope recognition:

  • Cusabio's antibody uses a synthesized peptide based on human HSP90AB1

  • Proteintech's 80301-1-RR employs a fusion protein (Ag1947)

  • OriGene's antibody is generated against full-length protein expressed in 293T cells

  • These differences affect epitope accessibility in various applications

• Antibody class and host species:

  • Choose between rabbit recombinant (80301-1-RR) and mouse monoclonal (67450-1-Ig, OTI4C10)

  • Consider compatibility with secondary detection systems and multiplex experiments

• Validation evidence:

  • Review Western blot data showing detection of the expected 83-90 kDa band

  • Evaluate knockout/knockdown validation data when available

  • Assess cross-reactivity profiles in relevant experimental systems

• Application-specific performance:

  • Some clones demonstrate superior performance in specific applications (e.g., 80301-1-RR for IF/ICC)

  • Consider whether the antibody has been validated in your specific application

The choice should ultimately be guided by your experimental design, target species, and application requirements, with preference given to antibodies with comprehensive validation data in systems similar to yours.

How can I optimize Western blot protocols for HSP90AB1 detection?

Optimizing Western blot protocols for HSP90AB1 detection requires attention to several technical parameters:

• Sample preparation:

  • Use standardized lysis buffers containing protease inhibitors to prevent degradation

  • Perform protein quantification and load equal amounts (typically 20-35μg of total protein)

  • Include positive controls from validated cell lines: HeLa, HEK-293, HepG2, Jurkat, NIH/3T3, or Neuro-2a cells

• Gel electrophoresis considerations:

  • Use 8-10% SDS-PAGE gels to achieve optimal resolution of HSP90AB1 (83-90 kDa)

  • Include molecular weight markers that span the 70-100 kDa range for accurate size determination

• Transfer optimization:

  • Employ wet transfer methods for larger proteins like HSP90AB1

  • Adjust transfer time and voltage based on protein size (typically longer transfers at lower voltage)

  • Verify transfer efficiency using reversible protein stains before immunodetection

• Blocking and antibody incubation:

  • Test different blocking agents (5% non-fat milk vs. BSA) to determine optimal signal-to-noise ratio

  • Dilute primary antibody according to manufacturer recommendations (ranging from 1:2000 for OriGene's OTI4C10 to 1:5000-1:50000 for Proteintech's antibodies)

  • Optimize incubation time and temperature (4°C overnight or room temperature for 1-2 hours)

• Detection system selection:

  • Choose detection systems based on desired sensitivity and dynamic range

  • Consider using enhanced chemiluminescence for routine detection

  • For quantitative analysis, fluorescent secondary antibodies may provide better linearity

Remember that HSP90AB1 exhibits slight molecular weight variations (83-90 kDa) depending on post-translational modifications and experimental conditions , which should be considered when interpreting results.

What are the recommended sample preparation methods for immunohistochemical detection of HSP90AB1?

For optimal immunohistochemical detection of HSP90AB1 in tissue samples, follow these methodological recommendations:

• Tissue fixation and processing:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Section tissues at 4-5μm thickness for optimal antibody penetration

• Antigen retrieval optimization:

  • Primary recommendation: Use TE buffer pH 9.0 for heat-induced epitope retrieval

  • Alternative method: Citrate buffer pH 6.0 may also be effective

  • Perform retrieval using pressure cooker, microwave, or water bath methods

  • Optimize retrieval time (typically 10-20 minutes) based on tissue type

• Blocking and antibody incubation:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide

  • Apply protein block to minimize non-specific binding

  • Dilute HSP90AB1 antibodies within recommended ranges:

    • CSB-RA904894A0HU: 1:50-1:200

    • 80301-1-RR: 1:250-1:1000

  • Incubate at 4°C overnight or at room temperature for 1-2 hours

• Detection system selection:

  • Use polymer-based detection systems for enhanced sensitivity

  • Select chromogen based on experimental needs (DAB for brightfield, fluorophores for multiplex)

  • Include hematoxylin counterstain for nuclear visualization in brightfield applications

• Controls and validation:

  • Include positive control tissue (human breast cancer tissue has been validated)

  • Implement negative controls (primary antibody omission)

  • Consider dual staining with other markers for colocalization studies

When troubleshooting, remember that optimal antibody concentration may vary depending on tissue type, fixation duration, and detection system sensitivity. Systematic optimization through dilution series testing is recommended for each new tissue type or experimental condition.

How does HSP90AB1 expression and function vary across different experimental models?

HSP90AB1 expression and function exhibit significant variability across experimental models, which researchers must consider when designing studies:

• Cell line expression profiles:

  • Validated expression in multiple cell types including HeLa, HEK-293, HepG2, Jurkat, NIH/3T3, Neuro-2a, HSC-T6, and PC-12 cells

  • Expression levels vary between cell types, with strongest Western blot signals typically observed in cancer cell lines

  • Consider endogenous expression levels when selecting appropriate models for functional studies

• Species-specific considerations:

  • High conservation across mammalian species enables cross-species reactivity of many antibodies

  • Zebrafish models show detectable HSP90AB1 expression, providing opportunities for developmental studies

  • Species-specific antibodies (e.g., human-specific CSB-RA904894A0HU ) should be selected when specificity is critical

• Tissue-specific expression patterns:

  • Differential expression observed across normal tissues

  • Elevated expression documented in pathological samples, particularly human breast cancer tissue

  • Consider tissue-specific post-translational modifications that may affect antibody recognition

• Functional variations in disease models:

  • In cancer models, HSP90AB1 interacts with and stabilizes oncogenic proteins

  • Stress response dynamics differ between normal and disease states

  • HSP90AB1 participates in various signaling pathways including NOD-like receptor signaling, cancer pathways, progesterone-mediated oocyte maturation, and prostate cancer signaling

• Experimental manipulation considerations:

  • Knockdown/knockout validation has been performed in select models

  • Consider inducible expression systems for temporal control of HSP90AB1 function

  • Evaluate stress-induced expression changes when designing experiments involving cellular stressors

Researchers should select experimental models based on both expression patterns and functional requirements, recognizing that observations in one model system may not directly translate to others.

What strategies can be employed to validate HSP90AB1 antibody specificity in experimental systems?

Rigorous validation of HSP90AB1 antibody specificity is essential for generating reliable research data. Consider implementing these comprehensive validation strategies:

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated knockout of HSP90AB1 provides the gold standard for specificity verification

  • siRNA or shRNA knockdown can demonstrate signal reduction proportional to knockdown efficiency

  • Overexpression systems using tagged HSP90AB1 constructs allow correlation between overexpression and antibody signal

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide/protein to block specific binding

  • Compare signals between blocked and unblocked antibody preparations

  • Signal abolishment indicates specificity for the target epitope

• Multiple antibody validation:

  • Compare signal patterns using antibodies targeting different HSP90AB1 epitopes

  • Consistent detection patterns across antibodies support specificity

  • Consider using both monoclonal (e.g., OTI4C10 ) and recombinant (e.g., 80301-1-RR ) antibodies

• Cross-species reactivity assessment:

  • Test antibody in species with known sequence homology to validate cross-reactivity claims

  • Compare detection in multiple reactive species (human, mouse, rat, zebrafish)

  • Confirm lack of signal in non-reactive species

• Application-specific validation:

  • For Western blot: Verify detection of the expected 83-90 kDa band

  • For IHC/IF: Confirm appropriate subcellular localization (primarily cytoplasmic)

  • For IP: Validate through mass spectrometry identification of immunoprecipitated proteins

• Biological validation:

  • Test antibody response to biological manipulations (heat shock, stress induction)

  • Verify expected changes in HSP90AB1 expression under conditions known to affect its levels

How can I design experiments to investigate HSP90AB1 interactions with client proteins?

Investigating HSP90AB1 interactions with client proteins requires sophisticated experimental approaches targeting protein-protein interactions:

• Immunoprecipitation (IP) strategies:

  • Use HSP90AB1 antibodies suitable for IP applications to pull down protein complexes

  • Perform reciprocal IPs using antibodies against suspected client proteins

  • Include appropriate controls (IgG control, input samples, knockout/knockdown validation)

  • Follow with Western blot detection or mass spectrometry analysis

• Proximity ligation assays (PLA):

  • Employ validated HSP90AB1 antibodies (e.g., 80301-1-RR for IF applications)

  • Combine with antibodies against putative client proteins

  • Visualize interactions as fluorescent puncta indicating proteins in close proximity (<40 nm)

  • Quantify interaction signals across experimental conditions

• Bimolecular fluorescence complementation (BiFC):

  • Generate fusion constructs of HSP90AB1 and client proteins with split fluorescent protein fragments

  • Co-express in appropriate cell models validated for HSP90AB1 expression

  • Analyze reconstituted fluorescence as indicator of protein-protein interactions

  • Include appropriate controls (non-interacting protein pairs)

• FRET/FLIM-based interaction studies:

  • Create fluorophore-tagged HSP90AB1 and client protein constructs

  • Express in relevant cell models (HeLa, HEK-293)

  • Measure energy transfer as indicator of protein proximity

  • Use photobleaching approaches to confirm specificity of interactions

• Pharmacological manipulation:

  • Employ HSP90 inhibitors to disrupt interactions

  • Monitor client protein stability/degradation following HSP90 inhibition

  • Correlate changes with biological outcomes in relevant models

• Stress-response dynamics:

  • Examine interaction patterns under various stress conditions (heat shock, oxidative stress)

  • Investigate temporal dynamics of interactions during stress response and recovery

  • Correlate with HSP90AB1's known role in stress response pathways

These approaches should be combined to provide complementary evidence for protein interactions, with particular attention to controls that rule out non-specific binding and artifacts.

What are common technical issues in HSP90AB1 antibody-based experiments and how can they be resolved?

Researchers frequently encounter technical challenges when working with HSP90AB1 antibodies. Here are common issues and evidence-based resolution strategies:

• Weak or absent Western blot signal:

  • Issue: Insufficient protein loaded or poor transfer efficiency

  • Resolution: Increase protein loading (35μg recommended) , optimize transfer parameters for high molecular weight proteins, and verify transfer efficiency with reversible staining

  • Mitigation: Use validated positive control cell lines (HeLa, HEK-293, HepG2)

• Non-specific bands in Western blot:

  • Issue: Suboptimal antibody dilution or cross-reactivity with related proteins

  • Resolution: Increase antibody dilution (test range from 1:5000-1:50000) , optimize blocking conditions, and include washing steps with higher stringency

  • Validation: Compare pattern with knockout/knockdown controls when available

• High background in IHC/IF applications:

  • Issue: Insufficient blocking or excessive antibody concentration

  • Resolution: Extend blocking time, test alternative blocking agents, and optimize antibody dilution (1:50-1:200 for IF/ICC, 1:250-1:1000 for IHC)

  • Technical adjustment: Use appropriate antigen retrieval methods (TE buffer pH 9.0 recommended)

• Inconsistent results between experiments:

  • Issue: Antibody degradation or variation in experimental conditions

  • Resolution: Aliquot antibodies upon receipt to avoid freeze-thaw cycles, store at -20°C as recommended , and standardize protocols

  • Quality control: Include internal controls in each experiment to normalize between runs

• Discrepancies in molecular weight:

  • Issue: HSP90AB1 observed between 83-90 kDa depending on experimental conditions

  • Resolution: Use appropriate molecular weight markers spanning 70-100 kDa range

  • Interpretation: Consider post-translational modifications that may alter migration patterns

• Poor signal in certain cell types:

  • Issue: Variable HSP90AB1 expression levels across cell types

  • Resolution: Adjust protein loading based on cell type, optimize antibody concentration for each model

  • Alternative: Consider more sensitive detection systems for cell types with lower expression

Systematic optimization and thorough documentation of experimental conditions are essential for troubleshooting HSP90AB1 antibody-based experiments and ensuring reproducible results.

How do I interpret differential HSP90AB1 expression patterns across experimental conditions?

Interpreting differential HSP90AB1 expression patterns requires careful consideration of both biological and technical factors:

• Quantification approaches:

  • Normalize HSP90AB1 signal to appropriate loading controls (β-actin, GAPDH, or total protein)

  • Employ densitometric analysis with appropriate software for Western blots

  • For IHC/IF, use quantitative image analysis measuring intensity, area, or H-score

  • Consider relative vs. absolute quantification methods based on experimental questions

• Statistical analysis considerations:

  • Perform experiments with sufficient biological replicates (minimum n=3)

  • Apply appropriate statistical tests based on data distribution

  • Consider fold-change and statistical significance when interpreting differences

  • Implement multiple comparison corrections for large-scale analyses

• Biological interpretation frameworks:

  • Consider HSP90AB1's role in stress response when analyzing stress-induced changes

  • Evaluate cell cycle effects, as HSP90AB1 participates in cell cycle progression pathways

  • Interpret cancer-related changes in context of HSP90AB1's interaction with oncogenic proteins

  • Assess correlation with client protein stability/function

• Technical validation of expression differences:

  • Confirm protein-level changes with mRNA expression when possible

  • Validate with alternative detection methods (e.g., IF to complement Western blot)

  • Consider antibody performance across the dynamic range of expression

• Contextual factors affecting interpretation:

  • Cell confluence effects on HSP90AB1 expression

  • Serum starvation or media composition influences

  • Post-translational modifications affecting antibody recognition

  • Subcellular localization changes not reflected in total protein measurements

When reporting differential expression, clearly describe normalization methods, quantification approaches, and statistical analyses to enable reproducibility and appropriate interpretation by the scientific community.

What considerations are important when designing multiplex experiments involving HSP90AB1?

Designing effective multiplex experiments involving HSP90AB1 requires careful consideration of multiple technical and biological parameters:

• Antibody compatibility assessment:

  • Select HSP90AB1 antibodies from different host species than other target antibodies

  • For immunofluorescence: Rabbit recombinant (80301-1-RR) can be paired with mouse antibodies

  • For Western blot: Consider using mouse monoclonal HSP90AB1 antibodies (67450-1-Ig, OTI4C10) with rabbit antibodies against other targets

  • Validate spectral compatibility of secondary antibody fluorophores or enzyme conjugates

• Technical optimization strategies:

  • Perform sequential immunostaining when using same-species antibodies

  • Optimize blocking steps to prevent cross-reactivity between detection systems

  • Consider tyramide signal amplification for sequential multiplex IHC

  • Test for antibody stripping efficiency if using sequential blotting approaches

• Experimental design considerations:

  • Include single-stained controls to establish baseline signals and spectral profiles

  • Implement appropriate negative controls for each antibody

  • Utilize spectral unmixing for fluorescence-based applications with overlapping emission spectra

  • Design image acquisition parameters to accommodate signal intensity ranges of all targets

• Biological relationship considerations:

  • Evaluate co-localization patterns between HSP90AB1 and client proteins or cofactors

  • Assess pathway activation markers alongside HSP90AB1 expression

  • Consider functional readouts in relation to HSP90AB1 expression/activity

  • Investigate temporal dynamics of multiple markers in stress response studies

• Data analysis approaches:

  • Employ co-localization analysis methods for IF/IHC multiplex experiments

  • Utilize multiparameter analysis to correlate HSP90AB1 with other markers

  • Consider machine learning approaches for complex pattern recognition

  • Implement appropriate statistical methods for multivariable analyses

When reporting multiplex experiments, provide detailed methodological descriptions including antibody combinations, detection systems, and analytical approaches to ensure reproducibility and appropriate interpretation of complex datasets.

How can HSP90AB1 antibodies be utilized in cancer research?

HSP90AB1 antibodies offer multiple applications in cancer research, reflecting the protein's role in oncogenic pathways:

• Biomarker studies:

  • Analyze HSP90AB1 expression across tumor types and stages using IHC (1:50-1:200 dilution)

  • Correlate expression with clinical outcomes and treatment response

  • Assess relationship with known cancer biomarkers in multiplex studies

  • Validated in human breast cancer tissue with appropriate antigen retrieval methods

• Therapeutic target validation:

  • Monitor HSP90AB1 expression/activity in response to HSP90 inhibitors

  • Evaluate effects on client protein stability using Western blot (1:5000-1:50000 dilution)

  • Assess combination therapy effects on HSP90AB1-dependent pathways

  • Investigate resistance mechanisms through client protein analysis

• Pathway analysis applications:

  • Study HSP90AB1 involvement in "Pathways in cancer" and "Prostate cancer" signaling

  • Investigate interaction with oncogenic client proteins using co-immunoprecipitation

  • Analyze correlation between HSP90AB1 expression and activation of downstream effectors

  • Explore stress response mechanisms in cancer cells using IF/ICC (1:50-1:200 dilution)

• Drug discovery applications:

  • Screen compounds for effects on HSP90AB1 expression/function

  • Evaluate HSP90AB1-client protein interactions in high-throughput formats

  • Develop assays for HSP90AB1 activity modulation

  • Create cellular models with modified HSP90AB1 expression for screening platforms

• Personalized medicine approaches:

  • Stratify patient samples based on HSP90AB1 expression patterns

  • Correlate with response to HSP90 inhibitors or other targeted therapies

  • Develop companion diagnostic approaches using validated antibodies

  • Investigate tumor heterogeneity through single-cell analysis of HSP90AB1 expression

These applications leverage the well-documented involvement of HSP90AB1 in cancer pathways and its interaction with many oncogenic proteins, making it both a biomarker and a potential therapeutic target .

What methodological considerations are important when studying HSP90AB1 in stress response pathways?

Investigating HSP90AB1 in stress response pathways requires specialized methodological approaches:

• Stress induction protocols:

  • Heat shock: Standardize temperature, duration, and recovery periods

  • Oxidative stress: Define H₂O₂ or other oxidant concentrations and exposure times

  • Proteotoxic stress: Establish proteasome inhibitor concentrations and treatment schedules

  • Control for confounding variables (cell density, passage number, media composition)

• Temporal dynamics assessment:

  • Design time-course experiments capturing immediate and delayed responses

  • Implement synchronized cell populations for cell cycle-dependent analyses

  • Consider pulse-chase approaches to study protein stability under stress

  • Develop live-cell imaging protocols using fluorescently tagged HSP90AB1

• Quantification approaches:

  • Western blot analysis using validated antibodies (1:5000-1:50000 dilution)

  • Real-time quantitative PCR to correlate transcript and protein levels

  • Immunofluorescence for subcellular localization changes (1:50-1:200 dilution)

  • Flow cytometry for population-level analysis (1:50-1:200 dilution)

• Functional readouts:

  • Client protein stability assessment under stress conditions

  • Chaperone activity assays measuring protein aggregation

  • Cell viability and apoptosis markers correlation with HSP90AB1 function

  • Protein complex formation analysis using native gel electrophoresis

• Integration with global stress response:

  • Multiplex analysis with other heat shock proteins (HSP70, HSP40)

  • Correlation with stress-activated transcription factors (HSF1)

  • Assessment of post-translational modifications affecting HSP90AB1 function

  • Evaluation of stress granule formation in relation to HSP90AB1 localization

These methodological considerations should be tailored to specific research questions while maintaining standardized protocols to ensure reproducibility across experiments examining HSP90AB1's role in cellular stress responses .

How can researchers effectively compare data generated using different HSP90AB1 antibody clones?

• Cross-validation experimental design:

  • Test multiple antibody clones on identical samples under standardized conditions

  • Include CSB-RA904894A0HU , 80301-1-RR , OTI4C10 , and 67450-1-Ig in parallel experiments

  • Maintain consistent protocols across antibodies, adjusting only dilution factors

  • Document epitope information and clone characteristics for proper interpretation

• Quantitative comparison methods:

  • Normalize signals to appropriate controls for each antibody

  • Consider relative rather than absolute quantification for cross-antibody comparisons

  • Implement correlation analyses to assess relationship between signals from different antibodies

  • Develop correction factors based on systematic comparative analyses

• Epitope-specific considerations:

  • Map epitope locations for each antibody when available

  • Consider post-translational modifications that might affect epitope recognition

  • Evaluate potential isoform-specific detection patterns

  • Assess potential masking of epitopes in protein complexes

• Technical normalization approaches:

  • Use standardized positive controls (HeLa, HEK-293 cell lysates)

  • Implement loading controls appropriate for each application

  • Consider spike-in standards for quantitative applications

  • Develop reference materials with known HSP90AB1 concentration

• Meta-analysis frameworks:

  • Document antibody-specific characteristics in publications (clone ID, dilution, protocol details)

  • Implement statistical methods appropriate for multi-antibody comparisons

  • Consider using effect sizes rather than absolute values when integrating data

  • Develop reporting standards for HSP90AB1 research to facilitate cross-study comparisons