HSP27 Antibody

What is HSP27 antibody and what are its typical research applications?

HSP27 antibody targets heat shock protein 27 (HSPB1), a small heat shock protein with a molecular mass of 27 kDa. This protein functions as a molecular chaperone in cells, assisting in refolding damaged proteins and preventing fat accumulation in certain pathways. Heat shock proteins are expressed in response to various stressful conditions including environmental, physical, and chemical stressors such as high temperature, viral infections, oxidative stress, and toxins .

The antibody is widely used in multiple research applications including:

• Western Blot (WB)

• Immunohistochemistry (IHC)

• Immunofluorescence (IF)/Immunocytochemistry (ICC)

• Flow Cytometry (FC)

• Immunoprecipitation (IP)

• Co-Immunoprecipitation (CoIP)

• ELISA assays

What are the recommended dilutions for HSP27 antibody across different experimental techniques?

The optimal dilution varies by application method and should be empirically determined for each experimental system. For reference, the following dilutions are recommended for antibody 18284-1-AP:

It is important to note that these dilutions may be sample-dependent, and researchers should titrate the reagent in each testing system to obtain optimal results .

What species reactivity has been confirmed for HSP27 antibody?

For the HSP27 antibody (18284-1-AP) specifically, tested reactivity has been confirmed with human, mouse, and rat samples. Cited reactivity in published literature extends to additional species including pig, chicken, and fish. This broad cross-species reactivity makes it a versatile tool for comparative studies across different model organisms .

How does in vivo inhibition of HSP27 affect immune responses to carcinogens?

In vivo inhibition of HSP27 (along with HSP70) produces significant immunological effects that impact carcinogenesis. Specifically, studies using C3H/HeN mice have demonstrated that this inhibition reduces T-cell mediated immune responses to carcinogens such as 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene B(a)P, resulting in antigen-specific tolerance .

The methodological approach involves:

• Pre-treatment of mice with anti-HSP27 and anti-HSP70 antibodies in vivo

• Subsequent exposure to a standard two-stage DMBA/12-O-tetradecanoylphorbol-13-acetate (TPA) cutaneous carcinogenesis protocol

• Monitoring tumor development and immune responses

Results show that antibody pre-treated animals developed significantly more tumors (p<0.05) compared to control groups. Mechanistically, mice pre-treated with these antibodies developed more H-ras mutations and fewer DMBA-specific cytotoxic T-lymphocytes, indicating that HSP27 plays a key role in cell-mediated immunity against carcinogenic polyaromatic hydrocarbons .

What methods can be used to assess HSP27-related cytotoxic T lymphocyte activity?

Cytotoxic T lymphocyte (CTL) activity related to HSP27 can be assessed using an in vivo antigen-specific cytotoxicity assay. The protocol involves:

• Topical treatment of mice with HSP27 and HSP70 antibodies

• Sensitization at the same site with DMBA

• Eight days post-sensitization, intravenous injection of 5 × 10^7 target cells

• Measurement of CTL-mediated cytotoxicity against these target cells

• Comparative analysis between antibody-treated and control groups

This methodology allows for the quantification of HSP27's impact on the development of antigen-specific cytotoxic T lymphocytes, which is crucial for understanding its role in immune surveillance of potentially cancerous cells.

How does HSP27 inhibition contribute to immune tolerance against carcinogens?

Research has demonstrated that inhibition of HSP27 using antibodies can induce immune tolerance to carcinogens like DMBA. The experimental evidence comes from a tolerance induction protocol:

• Pre-treatment of skin with anti-HSP antibodies (HSP27 and HSP70)

• Application of DMBA to the antibody-treated skin

• After a 14-day resting period, re-sensitization with 0.1% DMBA at a non-antibody treated site

• Ear challenge with DMBA 5 days after re-sensitization

• Measurement of ear swelling response as an indicator of immune reaction

The ear swelling response of mice pretreated with anti-HSP antibodies was suppressed compared to positive controls, despite the second sensitization attempt being through normal skin. This indicates that mice treated with anti-HSP antibody followed by carcinogen application became immunologically tolerant to DMBA .

What factors are associated with serum anti-HSP27 antibody titers based on machine learning approaches?

Advanced machine learning techniques have identified multiple factors associated with serum anti-HSP27 antibody titers. Using LightGBM model (with performance metrics RMSE: 0.1900 ± 0.0124; MAE: 0.1471 ± 0.0044; MAPE: 0.8027 ± 0.064 as mean ± sd) and SHAP (SHapley Additive exPlanations) method, researchers have identified these key factors in order of importance:

• Pro-oxidant-antioxidant balance (PAB) - showing the strongest association with a direct relationship

• Physical activity level (PAL) - with an indirect relationship

• Platelet distribution width (PDW)

• Mid-upper arm circumference (MUAC)

• Systolic blood pressure (SBP)

• Age

• Red cell distribution width (RDW)

• Waist-to-hip ratio (WHR)

• Neutrophils to lymphocytes ratio (NL)

• Platelet count (PLT)

• Serum glucose

• Serum cholesterol

• Red blood cells (RBC)

These findings enhance our understanding of the complex relationships between various physiological factors and anti-HSP27 antibody titers.

How can machine learning techniques improve anti-HSP27 antibody titer prediction compared to traditional statistical methods?

• Better handling of non-normal data distributions commonly found in biological systems

• Capability to identify complex, non-linear relationships between variables

• Improved prediction accuracy through ensemble methods

The methodological approach involves:

• Implementation of ensemble machine learning methods (LightGBM, CatBoost, XGBoost, AdaBoost)

• Model evaluation using multiple metrics during K-Fold cross-validation

• Feature importance determination using Permutation Feature Importance (PFI)

• Explanation of predictions using SHAP (SHapley Additive exPlanations) plots

This approach has demonstrated superior performance in predicting anti-HSP27 antibody titers and identifying their relationships with various factors compared to traditional statistical methods. The PFI and SHAP analyses particularly help in understanding both the magnitude and direction (positive/negative) of each factor's impact on predictions .

What cell types and tissues have validated positive results with HSP27 antibody?

HSP27 antibody (18284-1-AP) has been validated in multiple cell types and tissues with positive results:

For IHC applications, antigen retrieval is recommended with TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative .

What are the optimal storage conditions for preserving HSP27 antibody activity?

To maintain optimal activity, HSP27 antibody should be stored at -20°C. Under these conditions, it remains stable for approximately one year after shipment. The standard formulation includes:

• Storage buffer: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Form: Liquid

• Purification method: Antigen affinity purification

To prevent activity loss due to repeated freeze-thaw cycles, it is strongly recommended to aliquot the antibody upon receipt .

How can the relationship between HSP27 expression and disease states be quantified?

Quantifying the relationship between HSP27 expression and disease states involves several methodological approaches:

• Serum antibody titer measurement: Using ELISA to quantify anti-HSP27 antibody levels in patient populations versus controls

• Tissue expression analysis: Using IHC, WB, or IF to compare HSP27 protein levels across normal and pathological tissues

• Correlation analysis: Relating HSP27 levels to clinical parameters, disease progression markers, and outcomes

• Advanced statistical modeling: Employing machine learning techniques to identify complex relationships between HSP27 expression and multiple disease-related variables

Studies have found significant correlations between anti-HSP27 antibody titers and various medical conditions, particularly cardiovascular diseases. The overexpression of HSP27 in the body can trigger autoimmune responses, with increased anti-HSP27 antibody production serving as an inflammatory marker .

What are the critical considerations for designing HSP27 knockdown/knockout experiments?

When designing HSP27 knockdown or knockout experiments, researchers should consider:

• Selection of appropriate model system: Different cell lines and animal models may show varying responses to HSP27 manipulation

• Verification of knockdown efficiency: Using multiple techniques (WB, qPCR) to confirm successful reduction of HSP27

• Timing of analysis: HSP27 plays roles in both acute stress response and chronic adaptation

• Compensatory mechanisms: Other small heat shock proteins may compensate for HSP27 loss

• Phenotypic analysis scope: Include analyses of cell survival, stress response, cytoskeleton organization, and immune function

Published knockdown/knockout studies have demonstrated that HSP27 inhibition can significantly impact immune responses to carcinogens and potentially increase susceptibility to certain types of cancer .

How is HSP27 antibody being used in cancer immunotherapy research?

HSP27 antibody is being used in cancer immunotherapy research in several innovative ways:

• As a tool to understand the role of HSP27 in tumor development and progression

• To identify potential therapeutic targets in the HSP27 pathway

• For developing strategies to enhance anti-tumor immune responses

Research has shown that HSP27 plays a key role in the induction of cell-mediated immunity to carcinogenic compounds. Studies indicate that bolstering immune responses to carcinogenic polyaromatic hydrocarbons, potentially through modulation of HSP27 activity, may be an effective method for cancer prevention .

Initial findings suggest that inhibition of HSP27 might actually suppress anti-tumor immunity in some contexts, as demonstrated by the reduced T-cell mediated immune response to carcinogens and increased tumor development in animal models treated with anti-HSP27 antibodies .

Human Translation Tip: Researchers should carefully consider the potential immunosuppressive effects of HSP27 inhibition when developing therapeutic strategies targeting this pathway.

What are the latest machine learning approaches for analyzing HSP27-related biomarker data?

The most advanced machine learning approaches for analyzing HSP27-related biomarker data include:

• Ensemble methods: LightGBM, CatBoost, XGBoost, and AdaBoost have shown superior performance in predicting anti-HSP27 antibody titers compared to traditional statistical approaches

• Explainable AI techniques: SHAP (SHapley Additive exPlanations) and Permutation Feature Importance (PFI) help determine both the magnitude and direction of features' impact on predictions

• Cross-validation strategies: K-Fold cross-validation ensures robust model performance assessment

• Feature selection methods: Identifying the most important variables associated with HSP27 expression or anti-HSP27 antibody titers

Recent research employed LightGBM model evaluation metrics (RMSE: 0.1900 ± 0.0124; MAE: 0.1471 ± 0.0044; MAPE: 0.8027 ± 0.064) to identify pro-oxidant-antioxidant balance (PAB) and physical activity level (PAL) as the most important factors associated with anti-HSP27 antibody titers .