GSTM3 Antibody

What is GSTM3 and what tissues predominantly express it?

GSTM3 (glutathione S-transferase mu 3) is a cytosolic enzyme involved in prostaglandin and leukotriene synthesis and in the metabolization of various compounds. It is predominantly expressed in testis and brain tissues, as confirmed by multiple antibody validation studies . The protein has a calculated molecular weight of 27 kDa, though it is typically observed at 27-29 kDa in experimental conditions .

GSTM3 belongs to the mu class of glutathione S-transferases and has various alternative names in scientific literature, including:

• Brain GST

• Brain type mu glutathione S transferase

• Glutathione S-transferase, Mu 3

• GST class-mu 3

• GSTM3-3

• hGSTM3-3

Experimental evidence indicates that GSTM3 protein is endogenously expressed in HEK293 cells but not in HL-1 cardiac muscle cells, making HEK293 cells stably expressing Nav1.5 channel a preferred model for certain GSTM3 studies .

What are the recommended applications for GSTM3 antibodies?

GSTM3 antibodies have been validated for multiple experimental applications, with specific recommended dilutions for optimal results:

Researchers should note that optimal dilutions may be sample-dependent and should be titrated in each testing system to obtain optimal results. Both polyclonal rabbit antibodies described in the search results (15214-1-AP and DF12408) show reactivity with human and mouse samples .

What buffers and conditions are recommended for GSTM3 antibody storage?

GSTM3 antibodies should be stored according to manufacturer specifications to maintain reactivity and specificity. Typical storage conditions include:

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

• Storage temperature: -20°C

• Stability: One year after shipment when properly stored

• Aliquoting: Generally unnecessary for -20°C storage

• Special considerations: Some formulations may contain 0.1% BSA (for 20μl sizes)

Proper storage is critical for maintaining antibody performance across applications and preventing degradation that could lead to non-specific binding or reduced sensitivity.

What antigen retrieval methods are recommended for GSTM3 immunohistochemistry?

For optimal detection of GSTM3 in tissue sections using immunohistochemistry, specific antigen retrieval methods are recommended:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

Proper antigen retrieval is crucial for unmasking epitopes that may be cross-linked or hidden during fixation processes, particularly for formalin-fixed, paraffin-embedded (FFPE) tissues. The selection between TE buffer and citrate buffer may depend on specific tissue types and fixation methods used in the experiment.

How can GSTM3 antibodies be utilized to study its role in Brugada syndrome?

Recent research has identified GSTM3 as a potential genetic modifier in Brugada syndrome (BrS), a cardiac arrhythmia disorder. GSTM3 antibodies can be instrumental in investigating this relationship through several methodological approaches:

• Western blot analysis using GSTM3 antibodies can quantify protein expression levels in cardiac tissue or cell models to correlate with BrS phenotypes.

• Immunofluorescence techniques can localize GSTM3 in cellular contexts, particularly in relation to cardiac sodium channels.

• Co-immunoprecipitation using GSTM3 antibodies can identify protein-protein interactions that may explain molecular mechanisms.

Research findings indicate that a deletion containing the eighth exon and transcription stop codon of GSTM3 was observed in 23.9% of BrS patients versus 0.8% of controls. Patients carrying this deletion had significantly higher rates of sudden cardiac arrest (OR: 3.18, P<0.001) and syncope (OR: 1.76, P=0.04) . GSTM3 down-regulation in an oxidative stress environment leads to significant decrease of cardiac sodium channel current amplitude, potentially explaining the phenotypic manifestations in BrS patients .

What experimental controls should be included when using GSTM3 antibodies?

When designing experiments with GSTM3 antibodies, several controls should be included to ensure reliable and interpretable results:

• Positive tissue controls: Mouse, human, or rat testis tissues have been validated as positive controls for GSTM3 expression . Mouse testis tissue specifically has been recommended as a positive control for Western blot validation .

• Positive cell line control: HEK293 cells have been demonstrated to endogenously express GSTM3 .

• Negative cell line control: HL-1 cardiac muscle cells do not express detectable levels of GSTM3 and can serve as negative controls .

• Knockdown/knockout validation: Several publications have utilized GSTM3 knockdown or knockout models to validate antibody specificity, including gstm3 knockout zebrafish which showed greater ventricular arrhythmia incidence at baseline and after flecainide treatment .

• Loading controls: Standard loading controls appropriate for the subcellular localization of GSTM3 (cytosolic protein) should be included in Western blot experiments.

Including these controls helps validate antibody specificity, ensure proper experimental technique, and provide context for interpreting results, particularly in disease model systems.

How can researchers troubleshoot non-specific binding with GSTM3 antibodies?

Non-specific binding is a common challenge when working with antibodies. For GSTM3 antibodies specifically, researchers can implement several strategies to minimize this issue:

• Optimize antibody concentration: Titrate the antibody using the recommended dilution ranges (e.g., 1:2000-1:12000 for Western blot) . Too high a concentration can lead to non-specific binding.

• Extend blocking step: Use 5% non-fat dry milk or BSA in TBST for at least 1 hour at room temperature to reduce non-specific binding sites.

• Increase washing stringency: Additional wash steps with TBST can help remove non-specifically bound antibody.

• Validate with genetic models: Confirm specificity using GSTM3 knockout or knockdown samples. Published applications with knockdown/knockout models can provide validation references .

• Evaluate cross-reactivity: Consider potential cross-reactivity with other GST family members, particularly other mu-class GSTs which share sequence homology. Sequence alignment analysis can help predict potential cross-reactivity.

• Secondary antibody controls: Include secondary-only controls to distinguish between non-specific binding of primary versus secondary antibodies.

When troubleshooting persistent non-specific binding issues, researchers may need to compare multiple GSTM3 antibodies from different sources or those targeting different epitopes within the protein.

What approaches can be used to study GSTM3 gene deletions in patient samples?

The identified GSTM3 deletion associated with Brugada syndrome requires specific methodological approaches for detection and analysis:

• PCR-based genotyping: Design primers specific to the region with the lowest copy number of GSTM3. For the deletion identified in BrS patients (chr1:109737011-109737301, hg38), specialized PCR protocols can be developed to specifically amplify this region .

• Copy number variation analysis: A CNV region can be defined as a deletion if its copy number is less than 1.2. This approach was used in genome-wide microarray studies of BrS patients .

• Validation using multiple platforms: To minimize false associations due to technical artifacts, researchers should validate results using different platforms such as PCR-based genotyping, Sanger sequencing, and microarray or whole-exome sequencing .

• Control population comparison: Comparing deletion frequencies between patient cohorts and multiple control populations helps establish significance. This can include:

  • Ancestral-matched in-house controls (e.g., individuals with no arrhythmia-related symptoms)

  • Local biobank data (e.g., Taiwan Biobank)

  • Public databases like gnomAD structural variants database

• Functional validation: Using GSTM3 antibodies to correlate protein expression with gene deletion status in patient-derived samples or model systems.

These approaches enable comprehensive analysis of GSTM3 genetic variations and their potential clinical significance in disease contexts.

What cell and tissue models are optimal for studying GSTM3 function?

Based on the search results, several cell and tissue models have been validated for GSTM3 research:

When selecting models for GSTM3 research, investigators should consider both the baseline expression level of GSTM3 and the suitability of the model for the specific research question. For cardiac-related studies, the combination of HEK293 cells expressing Nav1.5 channel and zebrafish models provides complementary in vitro and in vivo approaches .

How can researchers optimize Western blot protocols for GSTM3 detection?

Western blot is one of the most common applications for GSTM3 antibodies. For optimal results, researchers should consider these specific methodological recommendations:

• Sample preparation:

  • Protein lysate quantity: 50 μg of protein is recommended based on protocols used in published studies

  • Lysis buffer: Standard RIPA buffer with protease inhibitors is suitable for GSTM3 extraction

• Gel electrophoresis:

  • Expected molecular weight: Prepare gels that provide good resolution in the 27-29 kDa range

  • Positive control: Include mouse testis tissue as a validated positive control

• Antibody incubation:

  • Primary antibody dilution: 1:2000-1:12000 range, with titration recommended for each system

  • Incubation conditions: Typically overnight at 4°C for primary antibody

• Detection:

  • Enhanced chemiluminescence is an effective detection method as used in published protocols

  • Expected band size: 27-29 kDa

• Validation:

  • GSTM3 knockout or knockdown samples provide definitive controls for antibody specificity

  • Multiple publications have used GSTM3 antibodies in Western blot applications, providing reference data for comparison

Optimization of these parameters will help ensure specific detection of GSTM3 protein and minimize background issues that can complicate interpretation.

What are the key considerations for co-localization studies involving GSTM3?

Co-localization studies using immunofluorescence techniques can provide valuable insights into GSTM3 function and interactions. Key methodological considerations include:

• Cell type selection:

  • HepG2 cells have been validated for IF/ICC applications with GSTM3 antibodies

  • For cardiac studies, HEK293 cells expressing Nav1.5 channels would be appropriate given GSTM3's implication in cardiac function

• Antibody optimization:

  • Recommended dilution range for IF/ICC: 1:200-1:800

  • Verify specificity using siRNA knockdown controls

• Co-staining considerations:

  • Select complementary fluorophores with minimal spectral overlap

  • When co-staining with other GST family members, careful antibody selection is necessary to avoid cross-reactivity

  • For cardiac sodium channel co-localization studies, Nav1.5-specific antibodies with validated specificity should be used

• Image acquisition:

  • Confocal microscopy is preferred for precise co-localization assessment

  • Include single-stained controls for accurate fluorophore compensation

• Quantitative analysis:

  • Employ co-localization coefficients (e.g., Pearson's correlation coefficient, Manders' overlap coefficient)

  • Consider super-resolution microscopy techniques for detailed subcellular localization

These methodological considerations help ensure reliable and interpretable results from co-localization experiments involving GSTM3.