HSP27 Mouse

What is HSP27 and what is its significance in research?

HSP27 is a small heat shock protein with a molecular weight of approximately 27 kDa that plays crucial roles in cellular stress response. Research significance includes its upregulation during ischemia/reperfusion injury, involvement in neurodegenerative diseases like ALS, and implications in cancer progression . The protein's oligomerization pattern is governed by its phosphorylation state, which may influence its protective capabilities against various cellular stresses . Beyond its chaperone functions, HSP27 has been identified as an estrogen-regulated protein that is identical to the estrogen-induced p29 and 24K protein, with approximately 50% of breast carcinomas showing HSP27 positivity, particularly those that are also positive for estrogen and/or progesterone receptors .

What are the general characteristics of mouse monoclonal antibodies against HSP27?

Mouse monoclonal antibodies against HSP27 typically recognize a 27 kDa protein band in Western blot applications . Most commercially available mouse monoclonal antibodies against human HSP27 do not cross-react with rodent HSP27, making them suitable for detecting human HSP27 in transfected rodent cells or human samples but not endogenous mouse HSP27 . These antibodies are commonly generated using recombinant full-length human HSP27 expressed in and purified from E. coli as the immunogen . They are typically available in various formats including unconjugated or conjugated to fluorescent dyes for different applications.

What applications are mouse anti-HSP27 antibodies suitable for?

Mouse anti-HSP27 antibodies are suitable for multiple research applications with specific recommended dilutions:

Optimization of dilutions for specific experimental conditions is recommended as sensitivity may vary between antibody clones and experimental systems .

How does HSP27 expression change in ALS mouse models?

In transgenic mutant SOD1 (G93A) mouse models of ALS, HSP27 shows distinct expression patterns that evolve with disease progression:

This changing pattern of expression and localization suggests HSP27 may play different roles as ALS progresses . Immunoblotting confirms that HSP27 is present in the nuclear-enriched fraction of spinal cord tissue from presymptomatic through advanced symptomatic stages, while significant cytoplasmic upregulation only becomes evident at later disease stages .

What is the significance of HSP27 nuclear localization in neurodegenerative disease models?

Nuclear localization of HSP27 in early stages of neurodegenerative disease models may represent an initial protective response. In ALS mouse models, HSP27 is clearly present in the nuclear-enriched fraction from presymptomatic through advanced symptomatic animals, while in wild-type animals, HSP27 is hardly detectable in the nuclear fraction . This nuclear accumulation precedes cytoplasmic upregulation, suggesting a sequential response to cellular stress. The nuclear localization may be associated with protection of nuclear proteins and DNA, transcriptional regulation, or modulation of apoptotic pathways. Double immunostaining for HSP27 and GFAP demonstrates that the nuclear HSP27 localization occurs in both neurons and glial cells, potentially indicating cell type-specific functions in the disease process .

How do transgenic HSP27 mouse models differ from endogenous HSP27 studies?

Transgenic mice overexpressing human HSP27 serve as valuable models for studying protective mechanisms against various stressors. Research demonstrates that transgenic mice overexpressing wild-type human HSP27 show different responses to stressors compared to those expressing mutant HSP27 variants . These models allow for the evaluation of how HSP27 phosphorylation and oligomerization states affect protection against cellular stresses like ischemia/reperfusion injury.

When designing experiments with these models, researchers should consider:

• Human and mouse HSP27 sequence differences that may affect protein-protein interactions

• Interpretation challenges when antibodies react only with human HSP27 but not mouse HSP27

• Potential non-physiological effects of transgenic overexpression

• The need for appropriate controls including wild-type mice

What are the optimal methods for subcellular fractionation to study HSP27 localization?

For studying HSP27's nuclear versus cytoplasmic distribution:

• Tissue homogenization: Use appropriate buffer containing sucrose, HEPES, KCl, and MgCl₂

• Differential centrifugation: Separate nuclei from cytoplasmic components

• Fraction verification: Use nuclear markers (e.g., histone H3) and cytoplasmic markers (e.g., GAPDH)

• Nuclear extraction: Apply specific nuclear lysis buffers containing salt and detergents

• Western blotting: Analyze equal protein amounts from each fraction using anti-HSP27 antibodies

• Quantification: Apply densitometry to determine relative distribution between compartments

This approach has successfully demonstrated HSP27 nuclear accumulation in ALS mouse models prior to significant cytoplasmic upregulation .

How should phosphorylation-specific analysis of HSP27 be performed?

To analyze HSP27 phosphorylation status:

• Sample preparation: Include phosphatase inhibitors to preserve in vivo phosphorylation state

• Phospho-specific antibodies: Use antibodies recognizing specific phosphorylation sites (Ser15, Ser78, Ser82)

• 2D electrophoresis: Separate phosphorylated isoforms based on charge differences

• Phos-tag gels: Apply this specialized acrylamide for enhanced separation of phosphorylated forms

• Mass spectrometry: For comprehensive phosphorylation site mapping and quantification

• Functional correlation: Compare phosphorylation patterns with oligomerization state and protective functions

These approaches are particularly relevant when studying HSP27's role in stress responses, as phosphorylation governs oligomerization patterns that influence protective capabilities .

What are the recommended immunohistochemistry protocols for HSP27 detection?

For optimal HSP27 detection in tissue sections:

• Fixation: Use 4% paraformaldehyde for 24 hours followed by paraffin embedding

• Sectioning: Prepare 5-7 μm sections mounted on positively charged slides

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0)

• Blocking: Apply 5-10% normal serum from secondary antibody species plus 0.1% Triton X-100

• Primary antibody: Incubate with anti-HSP27 antibody at appropriate dilution (approximately 1:50 for IHC)

• Secondary detection: Use biotin-streptavidin or polymer-based detection systems

• Double labeling: For cell-type identification, perform sequential or simultaneous staining with markers like GFAP

• Controls: Include sections from tissues known to be negative for HSP27

This protocol has been successfully used to demonstrate the nuclear localization of HSP27 in neurons and glial cells in ALS mouse models .

How can HSP27 mouse models be used to study ischemia/reperfusion injury protection?

HSP27 has shown protective effects against ischemia/reperfusion (I/R) injury, particularly in cardiac tissue. Transgenic mice overexpressing wild-type human HSP27 or mutant HSP27 variants can be used to investigate protective mechanisms . Research approaches include:

• Surgical models: Create controlled I/R injury in transgenic versus wild-type mice

• Functional assessment: Measure tissue damage, functional recovery, and molecular markers of injury

• Comparative analysis: Evaluate differences between wild-type HSP27 and phosphorylation-site mutants

• Mechanistic studies: Determine if protection correlates with oligomerization state, which is governed by phosphorylation

• Therapeutic implications: Test pharmacological approaches that modulate HSP27 phosphorylation

These approaches help elucidate how HSP27's oligomerization, which is regulated by phosphorylation, contributes to cardioprotection against I/R injury .

What are the considerations when studying HSP27 in cancer models?

HSP27 has been implicated in cancer progression and drug resistance, with approximately 50% of breast carcinomas showing HSP27 positivity . Key research considerations include:

• Model selection: Choose appropriate cancer cell lines or mouse models with differential HSP27 expression

• Expression modulation: Use knockdown or overexpression approaches to manipulate HSP27 levels

• Drug response testing: Evaluate chemotherapeutic sensitivity in relation to HSP27 status

• Hormone receptor correlation: Assess relationship between HSP27 and estrogen/progesterone receptor status

• Biomarker potential: Evaluate HSP27 as a prognostic or predictive marker

• Therapeutic targeting: Test HSP27 inhibitors alone or in combination with standard therapies

These approaches can help elucidate HSP27's role in cancer progression and drug resistance, potentially identifying new therapeutic strategies .

How to address species cross-reactivity issues with HSP27 antibodies?

Most mouse monoclonal antibodies against human HSP27 do not cross-react with rodent HSP27, presenting challenges for researchers . Strategies to address this include:

• Antibody selection: Carefully review manufacturer specifications for species reactivity (human vs. rodent)

• Validation: Confirm antibody specificity using positive and negative controls

• Alternative approaches: Consider rabbit polyclonal antibodies which may offer broader species cross-reactivity

• Recombinant systems: Use tagged HSP27 constructs when studying specific domains or mutations

• Species-specific primers: For mRNA expression studies, design primers that distinguish human from mouse HSP27

• Transgenic models: When using human HSP27 transgenic mice, select antibodies that specifically detect human HSP27

Understanding these limitations is crucial when designing experiments and interpreting results involving HSP27 detection in mouse models .

What are the common pitfalls in HSP27 oligomerization studies?

Studying HSP27 oligomerization presents several technical challenges:

• Sample preparation: Native conditions must be maintained to preserve oligomeric structures

• Size determination: Size exclusion chromatography requires careful calibration for accurate molecular weight estimation

• Dynamic equilibrium: HSP27 oligomers exist in dynamic equilibrium affected by concentration and buffer conditions

• Phosphorylation effects: Phosphorylation status dramatically influences oligomerization patterns

• Temperature sensitivity: Storage and handling temperatures can alter oligomeric distribution

• Concentration dependence: Protein concentration influences oligomerization equilibrium

• Detection limitations: Some methods may not capture the full range of oligomeric species

Addressing these challenges requires careful experimental design and multiple complementary approaches to accurately characterize HSP27 oligomeric states, which are critical for understanding its function in stress protection .