HSPB11 Antibody

What is HSPB11 and why is it important in biomedical research?

Research has established that HSPB11 is involved in several oncogenic processes, including:

• Cell cycle checkpoints

• G2M checkpoint regulation

• E2F target activation

• Rho GTPase signaling

• KRAS signaling pathways

Additionally, HSPB11 expression has been linked to immune cell infiltration in tumors, particularly affecting Th2 cells and dendritic cells, suggesting a role in cancer immunoregulation .

What molecular characteristics of HSPB11 are important for antibody selection?

When selecting HSPB11 antibodies for research, consider these key molecular characteristics:

For phosphorylation studies, it's important to note that in Chlamydomonas, HspB11 has been shown to be a phosphoprotein, with two predicted phosphorylation sites conserved between Chlamydomonas and rat .

What are the optimal conditions for Western blot detection of HSPB11?

For successful Western blot detection of HSPB11, follow these methodological guidelines:

For phosphorylation studies, use phosphatase inhibitors during sample preparation to preserve phosphorylated forms of HSPB11, which may appear as a higher molecular weight band in a doublet pattern .

How should HSPB11 antibodies be optimized for immunohistochemistry applications?

For optimal immunohistochemical detection of HSPB11 in tissue samples:

When scoring HSPB11 expression in HCC tissues, consider that HSPB11 overexpression correlates with advanced tumor stage, with poorer tumor differentiation showing higher expression levels .

How can HSPB11 antibodies be used to investigate its role in cancer progression?

To investigate HSPB11's role in cancer progression, implement these methodological approaches:

• Expression Analysis in Clinical Samples:

  • Compare HSPB11 levels in tumor vs. adjacent normal tissues using IHC and WB

  • Correlate expression with clinical parameters (stage, grade, survival)

  • Create prognostic models incorporating HSPB11 expression data

• Functional Studies:

  • Use siRNA or CRISPR to knockdown/knockout HSPB11 in cancer cell lines

  • Assess effects on proliferation, migration, invasion, and colony formation

  • Evaluate changes in cell cycle distribution and apoptosis rates

• Signaling Pathway Analysis:

  • Investigate HSPB11's involvement in key oncogenic pathways:

    • G2M checkpoint

    • E2F targets

    • Mitotic spindle assembly

    • KRAS signaling

    • Rho GTPase signaling

• Immune Cell Interaction Studies:

  • Use dual immunostaining to assess HSPB11 expression relative to immune cell markers

  • Perform co-culture experiments with immune cells and cancer cells with modulated HSPB11 expression

  • Evaluate cytokine profiles in the presence/absence of HSPB11

Research has demonstrated that HSPB11 expression correlates positively with Th2 cell abundance and negatively with dendritic cell levels, suggesting HSPB11 may promote an immunosuppressive tumor microenvironment .

What methodologies are recommended for studying HSPB11's role in intraflagellar transport?

To investigate HSPB11's function in intraflagellar transport and ciliary signaling:

• Localization Studies:

  • Use immunofluorescence with HSPB11 antibodies to visualize ciliary distribution

  • Perform live-cell imaging with fluorescently tagged HSPB11

  • Conduct super-resolution microscopy to precisely locate HSPB11 within the cilium

• Protein-Protein Interaction Analysis:

  • Identify HSPB11 binding partners through co-immunoprecipitation and mass spectrometry

  • Validate interactions using proximity ligation assays or FRET

  • Map interaction domains through deletion or mutation constructs

• Functional Assessment:

  • Generate HSPB11 knockdown/knockout cells and assess ciliary formation and function

  • Evaluate Hedgehog signaling pathway activity using reporter assays

  • Analyze ciliary protein transport using photoactivatable or pulse-chase approaches

• Phosphorylation Studies:

  • Use phospho-specific antibodies to detect HSPB11 phosphorylation states

  • Generate phosphomimetic or phospho-deficient HSPB11 mutants

  • Assess how phosphorylation affects HSPB11's interaction with other IFT components

Research has indicated that HSPB11 is not required for ciliary assembly but is essential for proper Hedgehog signaling, which occurs within cilia. Cilia lacking HSPB11 have defects in signal-dependent transport of Hedgehog components and fail to activate the pathway upon stimulation .

How can researchers validate the specificity of HSPB11 antibodies?

Comprehensive validation of HSPB11 antibodies should include multiple approaches:

• Genetic Validation:

  • Test antibody reactivity in HSPB11 knockout/knockdown models

  • Compare signal between wild-type and HSPB11-deficient samples

  • Validation score improves with demonstrated signal reduction in knockout samples

• Recombinant Expression Validation:

  • Overexpress HSPB11 in a cell line with low endogenous expression

  • Compare antibody reactivity between control and overexpressing cells

  • Look for increased signal intensity in overexpressing samples

• Independent Antibody Validation:

  • Compare staining patterns of multiple antibodies targeting different HSPB11 epitopes

  • Concordant results from independent antibodies increase confidence in specificity

• Orthogonal Validation:

  • Correlate protein detection with mRNA expression data

  • Compare antibody results with other protein detection methods

• Molecular Weight Verification:

  • Confirm detection at the expected molecular weight (16-20 kDa)

  • Be aware that HSPB11 may appear as a doublet due to phosphorylation

The Human Protein Atlas provides validation data for several HSPB11 antibodies, including enhanced validation using recombinant expression techniques .

What are common issues when working with HSPB11 antibodies and how can they be resolved?

For phosphorylation studies, note that HspB11 appears as a doublet in some Western blots, with the upper band representing the phosphorylated form. Two phosphorylation sites are conserved between Chlamydomonas and rat, suggesting similar regulation across species .

How can HSPB11 antibodies be utilized in studying cancer immunoregulation?

To investigate HSPB11's role in cancer immunoregulation, consider these methodological approaches:

• Tumor Microenvironment Analysis:

  • Use multiplex immunofluorescence to simultaneously detect HSPB11 and immune cell markers

  • Quantify spatial relationships between HSPB11-expressing cells and immune populations

  • Correlate HSPB11 expression with immune cell infiltration patterns

• Mechanistic Studies:

  • Investigate how HSPB11 modulates:

    • Th2 cell recruitment and activation (positive correlation observed)

    • Dendritic cell function and maturation (negative correlation observed)

    • Cytokine production and signaling in the tumor microenvironment

• Therapeutic Targeting:

  • Develop and test HSPB11-targeting strategies to modulate immune responses

  • Evaluate combination approaches with immune checkpoint inhibitors

  • Assess changes in tumor immunogenicity after HSPB11 modulation

• Patient Stratification:

  • Use HSPB11 expression as a biomarker for immunotherapy response prediction

  • Develop scoring systems incorporating both HSPB11 and immune cell markers

  • Correlate HSPB11 levels with response to immunotherapeutic agents

Research has demonstrated that HSPB11 expression in HCC positively correlates with Th2 cell abundance (R = 0.426, P < 0.001) and negatively correlates with dendritic cell levels (R = −0.222, P < 0.001), suggesting HSPB11 may promote an immunosuppressive microenvironment favorable for tumor progression .

What are the latest methodological approaches for developing HSPB11 as a diagnostic biomarker?

For developing HSPB11 as a diagnostic biomarker, researchers should consider these advanced methodological approaches:

• Biomarker Qualification:

  • Assess HSPB11's discriminatory power between tumor and normal tissues (AUC = 0.923 reported for HCC)

  • Determine sensitivity and specificity across different cancer types and stages

  • Compare performance against established biomarkers

• Clinical Sample Analysis:

  • Develop standardized ELISA protocols for serum/plasma HSPB11 detection

  • Establish reference ranges in healthy controls and cancer patients

  • Evaluate pre-analytical variables affecting HSPB11 measurement

• Multimarker Panel Development:

  • Integrate HSPB11 with other biomarkers to improve diagnostic accuracy

  • Develop nomogram models incorporating HSPB11 with clinical parameters

  • Create and validate calibration models for survival prediction

• Antibody-Based Diagnostic Development:

  • Optimize antibody pairs for sandwich ELISA development

  • Evaluate detection limits and dynamic range (e.g., 0.094-10 ng/ml)

  • Assess antibody performance across diverse patient populations

Research has shown that combining HSPB11 expression with T stage and M stage in a nomogram model provides good predictive ability for 1-, 3-, and 5-year survival rates in HCC patients, demonstrating its potential as part of a prognostic assessment tool .

What factors should be considered when selecting between monoclonal and polyclonal HSPB11 antibodies?

Available monoclonal HSPB11 antibodies include Mouse Monoclonal 68059-3-PBS (validated for Cytometric bead array, Indirect ELISA) , while polyclonal options include Rabbit Polyclonal 15732-1-AP (validated for WB, IHC, IF, ELISA) .

How should researchers interpret phosphorylated versus non-phosphorylated HSPB11 in experimental data?

When analyzing HSPB11 phosphorylation data, consider these interpretive guidelines:

• Western Blot Interpretation:

  • HSPB11 may appear as a doublet, with the upper band representing phosphorylated protein

  • Quantify both bands separately to assess phosphorylation status

  • Use phosphatase treatment controls to confirm phosphorylation-dependent mobility shifts

• Physiological Significance:

  • Phosphorylation status may change in response to cellular stress conditions

  • In hyperosmotic stress studies, HSPB11 showed slight increases followed by decreases after 72 hours

  • Compare phosphorylation patterns across different experimental conditions

• Functional Analysis:

  • Assess how phosphorylation affects HSPB11's interaction with other IFT complex components

  • Evaluate the impact on ciliary transport function and Hedgehog signaling

  • Consider phosphorylation in the context of cancer progression and immune regulation

• Technical Considerations:

  • Preserve phosphorylation status during sample preparation by using phosphatase inhibitors

  • Consider phospho-specific antibodies for direct detection of phosphorylated sites

  • Use phosphomimetic (S→D) or phospho-deficient (S→A) mutants for functional studies

Importantly, two predicted phosphorylation sites are conserved between Chlamydomonas and rat, suggesting evolutionary conservation of this regulatory mechanism .