HSP90AB1 (Ab-254) Antibody

What experimental applications is HSP90AB1 (Ab-254) Antibody validated for?

The HSP90AB1 (Ab-254) Antibody has been validated for multiple applications including:

• ImmunoFluorescence (IF)

• Western Blot (WB)

• ELISA

• ImmunoHistoChemistry (IHC)

The recommended dilutions for optimal results are:

• Western Blot: 1:500-1:3000

• Immunohistochemistry: 1:50-1:100

These applications allow researchers to investigate HSP90AB1 expression, localization, and interactions across multiple experimental platforms.

What are the key structural features that distinguish HSP90AB1 from other HSP90 family proteins?

• Human HSP90AB1 has 724 amino acids versus HSP90AA1's 732 amino acids (or 723 and 731 respectively with N-terminal methionines removed)

• HSP90AB1 possesses a unique signature sequence LKID (residues 71-74) not present in other HSPs

• In HSP90AB1, the amino acids ESEDK are removed between phosphorylation sites that are present in HSP90AA1

• The amino acids TQTQDQPME at the N-terminal end of HSP90AA1 are replaced by VHHG in HSP90AB1

These structural differences may contribute to differential regulation and client protein specificity between HSP90 family members.

What is the biological function of HSP90AB1 and why is it relevant to disease research?

HSP90AB1 functions as an ATP-dependent molecular chaperone that:

• Participates in signal transduction, protein folding, and degradation

• Aids in the folding, stabilization, and modification of diverse protein substrates

• Collaborates with various co-chaperones to manage ATPase-driven conformational changes

• Transports client proteins between the cytoplasm and nucleus

• Interacts with client proteins including kinases, ubiquitin ligases, and transcription factors

HSP90AB1 is relevant to disease research because:

• It exhibits high expression across multiple cancer types including lung, esophageal, gastric, breast, and colorectal cancer

• In lung adenocarcinoma, its overexpression results in imperfect clinical predictions

• It's implicated in neurodegenerative diseases including Alzheimer's disease

• It plays a role in inflammation and aging-related diseases

• Suppression of HSP90AB1 reduces the potential of endothelial cells to form tube structures, suggesting its importance in angiogenesis

How can HSP90AB1 (Ab-254) Antibody be used to investigate phosphorylation dynamics at Serine-254?

The HSP90AB1 (Ab-254) Antibody was raised against a synthetic non-phosphopeptide derived from human HSP90B around the phosphorylation site of serine 254 (V-G-S(p)-D-E) . To study the phosphorylation dynamics, researchers can:

• Use the HSP90AB1 (Ab-254) Antibody to immunoprecipitate total HSP90AB1, then probe with phospho-serine specific antibodies

• Compare immunoblots of samples treated with or without phosphatase inhibitors to preserve the phosphorylation state

• Design experiments with stimuli known to activate relevant kinases, then monitor temporal changes in HSP90AB1 phosphorylation

• Perform site-directed mutagenesis (S254A or S254D) to create phospho-dead or phospho-mimetic mutants for functional studies

• Use phospho-specific antibodies in parallel with the HSP90AB1 (Ab-254) Antibody to determine the ratio of phosphorylated to total protein

This methodological approach can reveal how phosphorylation at Ser-254 regulates HSP90AB1 function in different cellular contexts.

What protocols are recommended for optimizing immunohistochemistry with HSP90AB1 (Ab-254) Antibody?

For optimal immunohistochemistry results with HSP90AB1 (Ab-254) Antibody:

• Tissue Preparation:

  • Use freshly prepared 10% neutral buffered formalin for fixation (12-24 hours)

  • Process and embed in paraffin following standard protocols

  • Section at 4-6 μm thickness

• Antigen Retrieval:

  • Test both citrate buffer (pH 6.0) and EDTA buffer (pH 9.0)

  • Heat treatment: 95-98°C for 15-20 minutes followed by cooling

• Blocking and Antibody Incubation:

  • Block with 3-5% normal serum (from secondary antibody species)

  • Use the recommended dilution (1:50-1:100)

  • Incubate at 4°C overnight for optimal signal-to-noise ratio

• Detection and Visualization:

  • Use polymer-based detection systems for improved sensitivity

  • DAB (3,3'-diaminobenzidine) as chromogen for permanent preparations

  • Include positive control (breast carcinoma tissue shows strong expression)

• Counterstaining:

  • Light hematoxylin counterstain to allow clear visualization of positively stained cells

The validation images show successful staining of human breast carcinoma tissue, which can serve as an appropriate positive control .

How can co-immunoprecipitation with HSP90AB1 (Ab-254) Antibody be used to identify novel client proteins and co-chaperones?

Co-immunoprecipitation (Co-IP) with HSP90AB1 (Ab-254) Antibody offers a powerful approach to identify novel client proteins and co-chaperones:

• Experimental Design:

  • Prepare lysates under non-denaturing conditions to preserve protein-protein interactions

  • Immunoprecipitate HSP90AB1 complexes using the antibody (typically 2-5 μg per 500 μg of protein lysate)

  • Analyze precipitated complexes by mass spectrometry or Western blotting

• Controls to Include:

  • IgG control: Use non-immune rabbit IgG to assess non-specific binding

  • Input control: Analyze pre-IP lysate to confirm target presence

  • Validation: Confirm known interactions with established HSP90AB1 partners

  • Competition assay: Pre-incubate antibody with immunizing peptide to demonstrate specificity

• Modifications for Different Research Questions:

  • To identify stress-responsive interactions: Compare normal vs. heat-shocked or drug-treated cells

  • To find disease-specific clients: Compare normal vs. cancer or neurodegenerative disease models

  • To identify ATP-dependent interactions: Include ATP vs. ADP in binding buffers

• Validation of Novel Interactions:

  • Reverse Co-IP with antibodies against the identified interacting proteins

  • Proximity ligation assays to confirm interactions in situ

  • Functional studies to determine biological relevance of the interaction

This approach has successfully identified numerous HSP90 client proteins involved in cancer, neurodegenerative diseases, and aging-related conditions .

What factors might cause inconsistent Western blot results with HSP90AB1 (Ab-254) Antibody?

When experiencing inconsistent Western blot results with this antibody, consider these common issues and solutions:

Optimizing these parameters should improve consistency and specificity of HSP90AB1 detection.

How can background staining be minimized in immunofluorescence studies with HSP90AB1 (Ab-254) Antibody?

To minimize background in immunofluorescence experiments:

• Fixation Optimization:

  • Test different fixatives (4% PFA, methanol, or acetone)

  • Optimize fixation time (typically 10-20 minutes for PFA)

  • Quench aldehyde groups with glycine (100 mM) or NH₄Cl (50 mM)

• Improved Blocking:

  • Extend blocking time (1-2 hours at room temperature)

  • Use 5-10% normal serum from secondary antibody species

  • Add 0.1-0.3% Triton X-100 for proper permeabilization

  • Include 1% BSA to reduce non-specific binding

• Antibody Optimization:

  • Titrate antibody beyond the recommended dilution range

  • Increase washing steps (5-6 times for 5 minutes each)

  • Centrifuge diluted antibody before use to remove aggregates

  • Incubate primary antibody at 4°C overnight instead of shorter room temperature incubation

• Autofluorescence Reduction:

  • Treat with 0.1% Sudan Black B in 70% ethanol after antibody incubation

  • Use specialized quenching reagents for tissues with high autofluorescence

  • Select fluorophores with emission spectra distinct from autofluorescence wavelengths

• Controls:

  • Secondary antibody only control to assess non-specific binding

  • Peptide competition control to verify specificity

  • Negative control tissues known to express low levels of HSP90AB1

These optimizations should significantly improve signal-to-noise ratio in immunofluorescence studies.

How can I distinguish between HSP90AB1 and other HSP90 isoforms in my experiments?

Distinguishing between HSP90 isoforms requires careful experimental design:

• Antibody Selection:

  • Use HSP90AB1 (Ab-254) Antibody which targets the unique region around Ser-254

  • Verify the antibody's specificity against the LKID sequence (residues 71-74) unique to HSP90AB1

• Molecular Techniques:

  • RT-qPCR with isoform-specific primers targeting unique regions

  • siRNA/shRNA knockdown specific to HSP90AB1 as validation control

  • Recombinant protein standards of different isoforms for size comparison

• Protein Separation:

  • Use high-resolution SDS-PAGE (8% gels) to separate HSP90AB1 (724 aa) from HSP90AA1 (732 aa)

  • Consider 2D electrophoresis to separate based on both molecular weight and isoelectric point

  • Employ Phos-tag™ gels to distinguish based on phosphorylation status

• Mass Spectrometry Approach:

  • Tryptic digestion followed by MS/MS analysis to identify isoform-specific peptides

  • Focus on regions with sequence divergence between isoforms

  • Quantify isoform ratios using label-free or labeled quantification methods

• Verification Strategy:

  • Compare staining patterns with multiple antibodies against different HSP90 isoforms

  • Use tissues or cell lines with known differential expression of HSP90 isoforms

  • Include genetic manipulation (overexpression, knockdown) as specificity controls

This multi-faceted approach enables reliable discrimination between HSP90AB1 and other HSP90 family members.

How can HSP90AB1 (Ab-254) Antibody be used to investigate the role of HSP90AB1 in cancer progression?

HSP90AB1 (Ab-254) Antibody can be employed in multiple strategies to investigate cancer progression:

• Expression Analysis Across Cancer Stages:

  • IHC on tissue microarrays containing primary tumors and metastatic lesions

  • Quantitative Western blot comparing expression across cell lines representing different stages

  • Correlation with clinical outcomes using patient-derived samples

• Subcellular Localization Studies:

  • Immunofluorescence to track potential translocation between cytoplasm and nucleus

  • Co-localization with client proteins relevant to cancer progression

  • Fractionation followed by Western blot to quantify compartment-specific distribution

• Functional Studies:

  • Immunoprecipitation to identify cancer-specific client proteins and co-chaperones

  • Analysis of phosphorylation status at Ser-254 in different tumor grades

  • Assessment of HSP90AB1-client protein interactions after treatment with HSP90 inhibitors

• Therapeutic Response Monitoring:

  • IHC of tumor biopsies before and after treatment with HSP90 inhibitors

  • Correlation between HSP90AB1 expression/phosphorylation and treatment resistance

  • Changes in client protein stability following therapeutic intervention

HSP90AB1 is overexpressed in multiple cancer types including lung, esophageal, gastric, breast, and colorectal cancer , making it a relevant target for cancer progression studies.

What approaches can be used to study HSP90AB1's role in neurodegenerative diseases?

To investigate HSP90AB1's role in neurodegenerative diseases:

• Colocalization Studies:

  • Double immunofluorescence with HSP90AB1 (Ab-254) Antibody and markers of protein aggregation (tau, β-amyloid, α-synuclein)

  • Confocal microscopy to assess spatial relationships with disease-specific inclusions

  • Quantification of colocalization in patient tissues versus controls

• Protein-Protein Interactions:

  • Co-immunoprecipitation to identify interactions with disease-associated proteins

  • Proximity ligation assays to visualize and quantify interactions in situ

  • Pull-down assays to determine direct binding to misfolded proteins

• Functional Intervention:

  • Analysis of HSP90AB1 expression after treatment with HSP90 inhibitors (e.g., 17-AAG)

  • Assessment of protein aggregation following HSP90AB1 knockdown/overexpression

  • Evaluation of neuronal function (electrophysiology, calcium imaging) in relation to HSP90AB1 modulation

• Animal Model Studies:

  • IHC for HSP90AB1 in transgenic disease models at different disease stages

  • Correlation between HSP90AB1 levels and disease progression

  • Therapeutic targeting of HSP90AB1 and assessment of cognitive/behavioral outcomes

Research has shown that in Alzheimer's disease, tau tangles and β-amyloid deposits colocalize with HSP90, and HSP90 plays a role in regulating their aggregation and degradation. HSP90 inhibitors like 17-AAG can mitigate β-amyloid-induced neurotoxicity .

How can HSP90AB1 (Ab-254) Antibody be used to investigate the relationship between HSP90AB1 and cellular senescence in aging research?

For aging and senescence research applications:

• Expression Analysis During Senescence:

  • Western blot to quantify HSP90AB1 levels in young versus senescent cells

  • IHC or IF to examine spatial distribution changes during senescence

  • Co-staining with senescence markers (p16, SA-β-gal) to correlate expression patterns

• HSF1-HSP90AB1 Regulatory Axis:

  • Chromatin immunoprecipitation to assess HSF1 binding to HSP90AB1 promoter

  • HSF1 knockdown/overexpression followed by HSP90AB1 expression analysis

  • Monitoring of the p38–NF-κB–SASP pathway components in relation to HSP90AB1 levels

• Phosphorylation Dynamics:

  • Analysis of Ser-254 phosphorylation status in young versus senescent cells

  • Investigation of AKT-HSP90AB1 interaction using co-IP before and after HSP90 inhibitor treatment

  • Assessment of phosphorylation-dependent client protein interactions

• Intervention Studies:

  • Titration of HSP90 inhibitors (e.g., 17-DMAG, GA) at different concentrations

  • Monitoring senescence markers after treatment

  • Comparing effects in different cell types and senescence models

Research indicates complex relationships between HSP90AB1 and senescence, with HSP90 inhibitors showing senolytic effects at specific concentrations. 17-DMAG has demonstrated the ability to enhance mouse health and longevity in aging models .

How should results from HSP90AB1 immunohistochemistry be quantified and interpreted in cancer tissues?

For rigorous quantification and interpretation of HSP90AB1 IHC in cancer tissues:

• Quantification Methods:

  • H-score: Multiply staining intensity (0-3) by percentage of positive cells (0-100) for score range 0-300

  • Allred score: Sum of proportion score (0-5) and intensity score (0-3) for range 0-8

  • Digital image analysis using specialized software for more objective assessment

• Pattern Analysis:

  • Subcellular localization (cytoplasmic, nuclear, or both)

  • Homogeneous versus heterogeneous expression within tumor

  • Expression at tumor margins versus tumor core

  • Stromal versus tumor cell expression

• Comparative Analysis:

  • Compare with matched normal tissue when available

  • Assess gradient changes from normal to dysplastic to malignant areas

  • Correlate with tumor grade, stage, and histological subtype

• Clinical Correlation:

  • Link expression patterns to clinical outcomes (survival, treatment response)

  • Consider HSP90AB1 expression in context of known prognostic markers

  • Analyze in relation to treatment history (pre- vs. post-therapy)

In lung adenocarcinoma, HSP90AB1 overexpression has been linked to imperfect clinical predictions , highlighting the importance of contextual interpretation.

How can contradictory results between different detection methods for HSP90AB1 be reconciled?

When faced with contradictory results across detection methods:

• Method-Specific Considerations:

  • Western blot: Detects denatured protein, may miss conformation-specific epitopes

  • IHC/IF: Preserves tissue architecture but may have epitope accessibility issues

  • ELISA: Quantitative but lacks spatial information

  • qPCR: Measures mRNA rather than protein levels

• Technical Resolution Strategies:

  • Use multiple antibodies targeting different HSP90AB1 epitopes

  • Compare different fixation methods for IHC/IF

  • Include appropriate controls for each technique

  • Optimize protocols for each specific application

• Biological Explanations for Discrepancies:

  • Post-translational modifications affecting epitope recognition

  • Protein-protein interactions masking antibody binding sites

  • Differential isoform expression across tissues

  • Subcellular compartmentalization affecting extraction efficiency

• Integrated Analysis Approach:

  • Weight results based on technical reliability of each method

  • Prioritize functional validation over pure detection

  • Consider cellular context and disease state

  • Use orthogonal techniques (genetic manipulation, activity assays)

This systematic approach helps resolve contradictions and develop a more complete understanding of HSP90AB1 biology in your specific research context.

What statistical approaches are recommended for analyzing HSP90AB1 expression data in relation to clinical outcomes?

For robust statistical analysis of HSP90AB1 expression in clinical studies:

These approaches provide rigorous assessment of HSP90AB1's clinical significance while accounting for the complexities of biological data.