HSP90B1 Antibody

What are the validated applications for HSP90B1 antibodies?

HSP90B1 antibodies have been successfully validated for multiple applications with specific optimal dilutions:

• Western Blot (WB): Most antibodies show strong detection at 1:500-1:16000 dilutions, with HSP90B1 typically appearing as a band at approximately 100 kDa .

• Immunohistochemistry (IHC): Effective at 1:20-1:200 dilutions, with suggested antigen retrieval using TE buffer pH 9.0 or citrate buffer pH 6.0 .

• Immunofluorescence (IF/ICC): Optimal dilutions range from 1:50-1:800 .

• Immunoprecipitation (IP): Typically 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate .

• Flow Cytometry: Approximately 0.40 μg per 10^6 cells in a 100 μl suspension .

Each application requires optimization in your specific experimental system, and antibody performance may vary between manufacturers.

What is the expected molecular weight of HSP90B1 in Western blot analysis?

The calculated molecular weight of HSP90B1 is 92 kDa, but the observed molecular weight in Western blot is typically 100-110 kDa . This discrepancy is common with HSP90B1 and represents post-translational modifications. Some specific observations include:

• Most research-validated antibodies detect HSP90B1 at approximately 100 kDa under reducing conditions .

• The mobility in SDS-PAGE can be affected by post-translational modifications, which may cause the observed band size to be inconsistent with the theoretical size .

• Using a knockout cell line as a negative control (such as HSP90B1 knockout HEK293T cells) is the strongest validation method to confirm specificity .

Which cells and tissues show reliable HSP90B1 expression for positive controls?

Based on validated Western blot and IHC results:

This data provides reliable positive controls for antibody validation and experimental design .

How can I validate the specificity of HSP90B1 antibodies in my experimental system?

A multi-faceted approach to validating HSP90B1 antibody specificity includes:

• Knockout/knockdown validation: Use HSP90B1 knockout or knockdown cell lines as negative controls. Multiple antibody suppliers have demonstrated specificity using HSP90B1 knockout HEK293T cell lines, showing band presence in parental lines and absence in knockout lines .

• Cross-reactivity testing: If working with non-human species, verify cross-reactivity. Many HSP90B1 antibodies are reactive with human, mouse, and rat samples due to high sequence conservation .

• Multiple antibody comparison: Use antibodies targeting different epitopes of HSP90B1 to confirm consistent patterns. Search results indicate antibodies targeting different regions (N-terminal domain, middle domain, and C-terminal domain) .

• Molecular weight verification: Confirm the detected band appears at approximately 100 kDa, which is the consistently observed molecular weight for HSP90B1 .

• Positive controls: Include known positive cell lines or tissues (see section 1.3) when validating a new antibody .

What are the optimal fixation conditions for HSP90B1 detection in immunohistochemistry and immunofluorescence?

For optimal HSP90B1 detection in fixed samples:

For IHC-P (paraffin-embedded sections):

• Immersion fixation in formalin is commonly used

• Antigen retrieval is critical, using either:

  • TE buffer at pH 9.0 (preferred method)

  • Citrate buffer at pH 6.0 (alternative method)

• Incubation with primary antibody:

  • Concentration: 1-20 μg/ml

  • Conditions: Either overnight at 4°C or 1-3 hours at room temperature

• Detection systems:

  • HRP-DAB (brown) with hematoxylin counterstain (blue) shows specific staining localized to cytoplasm and plasma membrane

For ICC/IF (immunocytochemistry/immunofluorescence):

• For cultured cells, immersion fixation is effective

• Clear staining has been demonstrated using 10 μg/ml of antibody for 3 hours at room temperature

• HSP90B1 typically localizes to the cytoplasm with an endoplasmic reticulum pattern

What are important considerations when studying HSP90B1 in different subcellular compartments?

HSP90B1 is primarily an endoplasmic reticulum (ER) resident protein, but its detection in different compartments requires specific considerations:

• Subcellular localization: Primarily in the ER lumen, but also identified in melanosomes (by mass spectrometry in melanosome fractions from stage I to stage IV) .

• Staining pattern expectations:

  • In immunofluorescence, expect predominantly cytoplasmic staining with an ER pattern

  • In IHC, specific staining is typically localized to cytoplasm and plasma membrane

• Co-localization studies: When examining potential localization outside the ER, co-staining with compartment-specific markers is essential:

  • ER markers (e.g., KDEL receptors)

  • Melanosome markers if studying melanocytes

  • Plasma membrane markers if examining surface expression

• Subcellular fractionation: When biochemically separating cellular compartments, use multiple markers to confirm fraction purity before HSP90B1 detection .

• Stress conditions: ER stress can affect HSP90B1 localization and levels, so controlling experimental stress conditions is important .

How should I design experiments to study HSP90B1's role in TLR signaling pathways?

Based on literature showing HSP90B1 as a critical chaperone for multiple TLRs, consider these methodological approaches:

• Cell models: Use B-cell-specific HSP90B1-null mice or cell lines with HSP90B1 knockout/knockdown. Research has shown that HSP90B1 ablation in B cells specifically affects TLR-mediated antibody production .

• Functional assays:

  • Measure TLR surface expression using flow cytometry with specific TLR antibodies (e.g., anti-TLR2-APC)

  • Assess TLR-mediated inflammatory responses following ligand stimulation

  • Evaluate antibody production following TLR stimulation, which is specifically attenuated in HSP90B1-deficient B cells

• Genetic approaches:

  • CRISPR/Cas9 gene editing has been successfully used to knock down HSP90B1 expression in human monocyte cell lines (U937)

  • Analyze HSP90B1 SNPs for association with TLR-mediated immune responses using both ex-vivo whole blood assays and intracellular cytokine staining

• Controls and comparisons:

  • Include both TLR-dependent and TLR-independent stimulation to demonstrate specificity

  • Compare HSP90B1's effect on multiple TLRs (research shows it chaperones TLR1, TLR2, TLR4, TLR5, TLR6, TLR7, and TLR9)

What experimental approaches are effective for studying HSP90B1's role in cancer progression?

Research has demonstrated HSP90B1 upregulation in various cancers and its involvement in tumor development:

• Expression analysis:

  • Quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry have been effective in detecting HSP90B1 overexpression in HNSC (head and neck squamous cell carcinoma) cell lines and tissues

  • Compare expression between tumor and adjacent non-tumorous tissues from patient cohorts

• Clinical correlation studies:

  • Correlate HSP90B1 expression with clinicopathological features (T-stage, M-stage, clinical stage)

  • Survival analysis using Kaplan-Meier plots to correlate expression with patient outcomes

• Functional assays:

  • Cell proliferation, migration, and invasion assays following HSP90B1 knockdown or overexpression

  • Apoptosis assessment using flow cytometry and Western blot detection of apoptotic markers (CASP3, BAX, BCL2)

• Pathway analysis:

  • Examine HSP90B1's effect on the PI3K/Akt/mTOR pathway, which mediates autophagy regulation in cancer cells

  • Assess changes in pathway components using Western blot after HSP90B1 modulation

• In vivo models:

  • Subcutaneous tumorigenic models and tail vein hematogenous metastasis models in mice using cell lines with stable HSP90B1 knockdown

  • Assess tumor size, growth rate, and metastatic potential

How does HSP90B1's function differ from cytosolic HSP90, and how should this influence experimental design?

Understanding the distinct properties of HSP90B1 (ER-resident) compared to cytosolic HSP90 is crucial for proper experimental design:

• Subcellular localization:

  • HSP90B1 is primarily localized in the ER lumen, while cytosolic HSP90 is found in the cytoplasm

  • Experiment design should include specific markers to distinguish between compartments in imaging studies

• Client protein specificity:

  • HSP90B1 has a distinct set of client proteins including TLRs and integrins, but notably NOT immunoglobulins

  • Cytosolic HSP90 has different client proteins

  • When studying chaperone-client interactions, consider this specificity

• Inhibitor selectivity:

  • Small molecule inhibitors like PU-H71 and geldanamycin have been used to study HSP90, but may have different affinities for HSP90B1

  • Chemical probes using immobilized inhibitors can enrich active, client-protein bound HSP90

  • Biotinylated geldanamycin (GA) with streptavidin beads can capture HSP90 and its binding partners, though with limited efficacy

• Conformational states:

  • HSP90B1 exists in different conformers and complexes

  • Research suggests a unique high-affinity conformation in cancer cells

  • Consider using methods that can distinguish between conformational states

• Cancer relevance:

  • Tumors can be classified into subtypes based on HSP90 connectivity

  • Type 1 tumors show connectivity between HSP90 and HSP70 chaperone systems and contain HSP90 species with pI >4.9

  • Type 2 tumors lack this connectivity

  • This distinction may explain varying results with HSP90 inhibitors in different cancer cells

How can I troubleshoot inconsistent Western blot results with HSP90B1 antibodies?

When facing inconsistent results in HSP90B1 Western blots, consider these methodological approaches:

• Sample preparation issues:

  • Ensure complete cell lysis using appropriate buffers (Immunoblot Buffer Group 1 has been effective)

  • Run samples under reducing conditions as validated in published protocols

  • Use fresh samples or properly stored ones, as protein degradation can affect results

• Antibody selection and optimization:

  • Optimal antibody concentrations typically range from 0.2-0.5 μg/mL for Western blot

  • Try antibodies targeting different epitopes if one region might be masked or modified

  • Use validated positive controls (see section 1.3)

• Detection challenges:

  • Expected molecular weight is approximately 100 kDa, though the theoretical size is 92 kDa

  • Include loading controls (GAPDH has been successfully used)

  • Consider the use of HRP-conjugated secondary antibodies specific to the host species of your primary antibody

• Validation methods:

  • Use HSP90B1 knockout cell lines as negative controls (such as HSP90B1 knockout HEK293T)

  • Compare results across multiple cell lines to establish consistency patterns

• Technical considerations:

  • PVDF membranes have been successfully used for HSP90B1 detection

  • Try different blocking solutions if background is an issue

  • Consider using a different detection system if sensitivity is a concern

What are the important considerations when designing HSP90B1 interactome studies?

HSP90B1 interactome studies present unique challenges due to the protein's abundance, ubiquity, and dynamic nature:

• Cell state considerations:

  • HSP90B1 is involved in stress responses, so the growth and physiological state of cells can heavily impact the interactome

  • Experimental conditions can easily influence these parameters

  • Standardize growth conditions and stress levels across experiments

• Subcellular fractionation:

  • Distinct interaction networks exist in particular subcellular locations

  • Careful fractionation with proper markers is essential to avoid contamination between compartments

• Capture methods:

  • Affinity pulldowns coupled to unbiased mass spectrometry (AP-MS) have been successful

  • LUminescence-based Mammalian IntERactome (LUMIER) technology is an alternative approach

  • Two-hybrid screens and chemical genetic interactions analysis can provide complementary data

• Affinity capture considerations:

  • Classical immunoprecipitation with specific antibodies

  • Affinity purification with bead-immobilized inhibitors like PU-H71

  • Tagged HSP90B1 capture methods

  • Each approach may favor certain conformers or complexes

• Chemical probes:

  • Small molecules (chemical probes) have been used to probe HSP90 function

  • Biotinylated geldanamycin (GA) with streptavidin beads has been used, though with limited efficacy

  • Immobilized inhibitors like PU-H71 can enrich the active, client-protein bound HSP90B1

• Analytical approaches:

  • Bioinformatics analysis of interaction networks is essential to interpret complex datasets

  • Validation of key interactions through biochemical methods is crucial

How should experimental approaches differ when studying HSP90B1 in different disease contexts?

Research approaches should be tailored to the specific disease context when studying HSP90B1:

• In cancer research:

  • Focus on proliferation, migration, invasion, and apoptosis assays

  • Examine the PI3K/Akt/mTOR pathway and autophagy regulation

  • Correlate expression with clinical staging and patient outcomes

  • Consider both in vitro cell line models and in vivo tumor models

• In immune system and inflammatory disorders:

  • Center on TLR function and signaling

  • Measure cytokine responses and inflammatory mediators

  • Evaluate B-cell functions including antibody production

  • Consider conditional knockout models (e.g., B-cell-specific HSP90B1-null mice)

• In infectious disease research:

  • Focus on TLR-mediated pathogen recognition

  • Study HSP90B1's role in mounting appropriate immune responses

  • Consider using microbial stimulation models to assess the impact on pathogen clearance

• In neurodegenerative diseases:

  • Emphasize protein folding and quality control functions

  • Study the unfolded protein response (UPR) in neuronal cells

  • Investigate potential interactions with disease-specific misfolded proteins

• In genetic association studies:

  • Screen for HSP90B1 SNPs associated with disease phenotypes

  • Evaluate functional consequences of genetic variants on HSP90B1 expression

  • Consider both ex-vivo assays and intracellular cytokine staining to assess functional impacts

Each research context requires specific controls, experimental readouts, and interpretation frameworks to properly establish HSP90B1's role in the disease process.

What are promising approaches to explore HSP90B1's role in cellular stress responses beyond current applications?

Several emerging techniques and approaches hold promise for advancing our understanding of HSP90B1:

• Single-cell analysis:

  • Single-cell proteomics to examine heterogeneity in HSP90B1 expression and function

  • Single-cell RNA-seq to identify transcriptional networks associated with HSP90B1 in stress responses

  • Spatial transcriptomics to map HSP90B1 activity in complex tissues

• Real-time imaging:

  • Live cell imaging using fluorescently tagged HSP90B1 to track dynamic responses to stress

  • FRET-based sensors to detect HSP90B1-client interactions in real time

  • Super-resolution microscopy to examine nanoscale organization of HSP90B1 complexes

• Integrated multi-omics:

  • Combining proteomics, transcriptomics, and metabolomics to create comprehensive models of HSP90B1 function

  • Network analysis approaches to identify key nodes in HSP90B1-dependent pathways

  • Machine learning algorithms to predict novel HSP90B1 functions from integrated datasets

• Therapeutic targeting:

  • Development of HSP90B1-specific inhibitors distinct from pan-HSP90 inhibitors

  • Exploration of client-specific disruption strategies

  • Investigation of combination approaches targeting HSP90B1 and complementary pathways

These approaches could significantly advance our understanding of HSP90B1's roles beyond current knowledge, particularly in stress response coordination, disease progression, and potential therapeutic applications.