GSR Antibody

What is GSR and why is it important in biological research?

GSR (Glutathione Reductase), also known as GLUR, GRD1, GR, or GRase, is an enzyme that maintains high levels of reduced glutathione in the cytosol . It belongs to the class-I pyridine nucleotide-disulfide oxidoreductase family . The GSH/GSSG redox couple is a critical intracellular determinant of antioxidant capacity because the abundance of GSH (~10–15 mM) is three to four orders of magnitude higher than other reductants such as NADPH and reduced thioredoxin . GSH can directly scavenge certain free radicals and reactive oxygen species (ROS), making the glutathione system one of the major defense mechanisms for cellular protection against oxidative stress . This enzyme's function is particularly significant in tissues with high metabolic activity where oxidative stress management is crucial, making GSR antibodies important tools for studying oxidative stress responses and antioxidant defense mechanisms.

What are the most common applications for GSR antibodies in research?

GSR antibodies are extensively validated for several key applications in research:

These applications enable researchers to study GSR expression patterns, protein-protein interactions, and localization in various cellular compartments and tissues. The versatility of GSR antibodies across these techniques allows for comprehensive analysis of this important enzyme in different experimental contexts, from basic expression studies to complex functional investigations .

How do I select the appropriate GSR antibody for my experiments?

When selecting a GSR antibody for your experiments, consider these critical factors:

First, determine your target species, as antibody reactivity varies. Available commercial GSR antibodies show reactivity with human, mouse, and rat samples . Second, identify which application is most appropriate for your research question - whether Western blot, immunohistochemistry, immunofluorescence, or immunoprecipitation. Each application requires specific antibody characteristics and optimization.

Third, consider the antibody type (monoclonal vs. polyclonal). Polyclonal antibodies like ab137513 and 18257-1-AP offer broader epitope recognition, while monoclonals provide more specificity . Fourth, verify the immunogen information - for example, the ab137513 antibody was raised against a recombinant fragment within human GSR amino acids 150-450 .

Finally, review validation data provided by manufacturers, including predicted band sizes (approximately 56 kDa for GSR) and validated protocols . Cell/tissue lysates that have been successfully used with the antibody (such as mouse brain lysate, HCT116 cells for ab137513, or HeLa cells and various tissues for 18257-1-AP) can guide your experimental design and provide appropriate positive controls .

What are the optimal conditions for Western blot analysis using GSR antibodies?

For optimal Western blot analysis with GSR antibodies, follow these methodological guidelines:

Sample preparation should include efficient protein extraction with protease inhibitors to prevent degradation. Based on validated protocols, prepare protein samples in standard SDS-PAGE loading buffer and denature at 95°C for 5 minutes. Load 30-50 μg of total protein per lane, as demonstrated in successful experiments with mouse brain lysate (50 μg) and HCT116 cell lysate (30 μg) .

For gel electrophoresis, use 10% SDS-PAGE gels, which have been successfully employed for GSR detection . After protein transfer to PVDF or nitrocellulose membranes, block with 5% non-fat milk or BSA in TBST buffer for 1 hour at room temperature.

The recommended antibody dilution ranges from 1:1000 to 1:4000 in blocking buffer . For example, ab137513 has been validated at 1:1000 dilution . Incubate membranes with primary antibody overnight at 4°C, followed by appropriate HRP-conjugated secondary antibody incubation (typically 1:5000-1:10000) for 1-2 hours at room temperature.

The expected molecular weight for GSR is approximately 56 kDa (predicted) , though observed molecular weights range between 52-55 kDa . Include appropriate positive controls such as Jurkat cells, HeLa cells, mouse lung tissue, or human placenta tissue, all of which have demonstrated positive GSR expression .

How should I optimize immunohistochemistry protocols for GSR detection in tissue samples?

For optimal immunohistochemistry (IHC) detection of GSR in tissue samples, follow these methodological recommendations:

Begin with proper tissue fixation, typically using 10% neutral buffered formalin, followed by paraffin embedding and sectioning at 4-6 μm thickness. Antigen retrieval is crucial for GSR detection - use TE buffer at pH 9.0 as recommended, though citrate buffer at pH 6.0 may serve as an alternative .

Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes, followed by protein blocking with 5-10% normal serum for 30-60 minutes at room temperature. For primary antibody incubation, use GSR antibody at dilutions between 1:200-1:800 , incubating overnight at 4°C in a humidified chamber.

After washing, apply appropriate biotinylated secondary antibody for 30-60 minutes at room temperature, followed by streptavidin-HRP conjugate. Develop signal using DAB substrate and counterstain with hematoxylin for nuclear visualization. Human breast cancer tissue has been validated as a positive control for GSR antibody in IHC applications .

For multiplex staining to co-localize GSR with other proteins, sequential staining protocols may be necessary, with careful selection of antibodies raised in different host species to avoid cross-reactivity. Always include appropriate negative controls by omitting primary antibody or using isotype control antibodies to confirm staining specificity.

What are the critical considerations for immunofluorescence detection of GSR?

For successful immunofluorescence (IF) detection of GSR, several critical considerations must be addressed:

Cell fixation method significantly impacts GSR epitope accessibility. While 4% paraformaldehyde (10-15 minutes at room temperature) is commonly used, methanol fixation (-20°C for 10 minutes) may better preserve GSR antigenicity for some antibodies. Permeabilization with 0.1-0.5% Triton X-100 for 5-10 minutes aids antibody access to intracellular GSR.

Optimal antibody dilution for IF applications ranges from 1:50 to 1:500, with validation confirmed in HeLa cells . Incubate primary antibody for 1-2 hours at room temperature or overnight at 4°C in a humidified chamber. Use appropriate fluorophore-conjugated secondary antibodies at 1:200-1:1000 dilutions with 1-hour incubation at room temperature, protected from light.

Since GSR functions in antioxidant defense, co-staining with mitochondrial markers (MitoTracker) or other redox-related proteins may provide valuable contextual information about cellular localization and functional associations. Always include DAPI or Hoechst staining for nuclear visualization to aid in subcellular localization assessment.

To minimize autofluorescence, particularly in tissues with high lipofuscin content, consider treating sections with Sudan Black B (0.1-0.3% in 70% ethanol) for 5-10 minutes before mounting. For mounting, use anti-fade mounting media containing DAPI to preserve fluorescence signal during imaging and storage.

How do I address non-specific binding or background issues with GSR antibodies?

Non-specific binding and background issues with GSR antibodies can be addressed through several methodological refinements:

For Western blots, if multiple bands appear beyond the expected 52-56 kDa band , increase blocking time to 2 hours using 5% BSA instead of milk, especially when phospho-specific antibodies are used concurrently. Increase washing duration and frequency (5 washes, 5 minutes each) with 0.1% Tween-20 in TBS. Further dilute primary antibody (try 1:5000 instead of 1:1000), and consider using different secondary antibodies with higher specificity.

For immunohistochemistry applications, high background may result from inadequate blocking or overly concentrated antibody. Implement a dual blocking strategy using 10% normal serum from the secondary antibody host species followed by 1% BSA. For human tissues, add human Fc receptor blocking reagent to prevent non-specific Fc receptor binding. Optimize antigen retrieval methods, as over-retrieval can increase background; test shorter retrieval times or lower temperatures.

For immunofluorescence, cellular autofluorescence can be distinguished from specific staining by examining unstained samples across different filter sets. Quench autofluorescence with 0.1% Sudan Black B treatment for 20 minutes or 50 mM NH₄Cl for 10 minutes before antibody application. Include appropriate negative controls for each experiment, including secondary-only controls and isotype controls, to distinguish true signal from background.

What are common pitfalls in GSR knockout/knockdown validation experiments?

When validating GSR knockout or knockdown experiments, researchers should be aware of several common pitfalls:

First, incomplete knockout verification is a major concern. Studies with Gsr knockout mice demonstrated significantly decreased GSR activity and GSH/GSSG ratios in the cytosol of inner ears, yet functional compensation through alternate pathways occurred . Always quantify knockout efficiency through multiple methods: Western blot for protein levels, qPCR for mRNA expression, and enzymatic activity assays for functional validation. Expect at least 80-90% reduction in targeted measures to consider knockdown effective.

Second, compensatory mechanisms can mask phenotypes. The thioredoxin system can support GSSG reduction in GSR-deficient models, as evidenced by increased activities of cytosolic thioredoxin and thioredoxin reductase in the inner ears of Gsr knockout mice . Examine expression and activity of functionally related proteins, including thioredoxin system components and other antioxidant enzymes, to identify potential compensatory mechanisms.

Third, background strain influences are critical. In GSR studies, mice were backcrossed onto the CBA/CaJ strain to eliminate confounding mutations (e.g., Cdh23) that could affect auditory phenotypes . Genomic validation of strain background, including sequencing of relevant loci like Cdh23 for auditory studies, may be necessary to interpret results correctly .

Finally, phenotypic effects may be context-dependent. While Gsr knockout mice showed no hearing deficits under normal conditions, phenotypes may only emerge under stress conditions, as observed with increased renal injury in Gsr-deficient mice exposed to diquat . Design experiments that include both baseline and stress conditions (oxidative challenge, nutrient deprivation, etc.) to fully characterize knockout phenotypes.

How do I interpret contradictory results between different detection methods for GSR?

When faced with contradictory results between different GSR detection methods, consider these methodological factors:

Epitope accessibility varies across techniques. Western blotting detects denatured proteins, exposing epitopes that might be masked in native conformations used in immunoprecipitation or immunofluorescence. If an antibody works in Western blot but not in IHC, the epitope may be masked by protein folding, post-translational modifications, or protein-protein interactions in fixed tissues. Try different fixation methods or antigen retrieval buffers (TE buffer pH 9.0 versus citrate buffer pH 6.0) to improve epitope accessibility .

GSR exists in multiple isoforms. Five isoforms are produced by alternative splicing and alternative initiation, and GSR can form homodimers with a molecular weight of 110 kDa . Antibodies targeting different epitopes may detect different isoforms, leading to apparent contradictions. Western blot may show bands at 52-56 kDa (monomeric forms) and/or at approximately 110 kDa (dimeric forms) . Compare your results with published molecular weights and determine which isoforms your antibody should detect based on the immunogen sequence.

Cross-reactivity with related proteins can cause false positives. GSR belongs to the pyridine nucleotide-disulfide oxidoreductase family , which includes other proteins with similar domains. Validate specificity using positive and negative controls, including samples with confirmed GSR expression (HeLa cells, Jurkat cells, mouse brain tissue) and, ideally, GSR knockout samples as negative controls.

Create a comprehensive validation table comparing results across methods, including antibody dilutions, detection protocols, and observed band patterns or staining distributions. This systematic approach will help identify method-specific factors contributing to contradictory results and guide methodology refinement.

How can GSR antibodies be utilized in studying redox balance and oxidative stress mechanisms?

GSR antibodies can be powerfully employed in studying redox balance and oxidative stress through several advanced approaches:

Co-immunoprecipitation with GSR antibodies can identify protein interaction partners under varying oxidative conditions. Using validated IP protocols with 0.5-4.0 μg antibody per 1.0-3.0 mg of protein lysate , researchers can pull down GSR complexes and identify associated proteins through mass spectrometry. This approach has revealed how GSR interacts with other redox-regulating proteins in stress response pathways.

Chromatin immunoprecipitation (ChIP) coupled with GSR antibodies can investigate potential non-canonical roles of GSR in transcriptional regulation during oxidative stress. While GSR is primarily cytosolic, evidence suggests some redox enzymes can translocate to the nucleus under stress conditions, potentially affecting gene expression patterns related to antioxidant response.

Proximity ligation assays using GSR antibodies with antibodies against potential interaction partners (thioredoxin, thioredoxin reductase) can visualize and quantify protein-protein interactions in situ. This technique is particularly valuable for studying how GSR interactions change dynamically during oxidative challenge, providing spatial and temporal resolution not possible with biochemical methods alone.

For in vivo studies, parallel analysis of GSR expression/localization alongside markers of oxidative damage (4-HNE, 8-OHdG) and antioxidant capacity (GSH/GSSG ratio) can correlate GSR regulation with functional outcomes. This multi-parameter approach has been valuable in understanding compensatory mechanisms in GSR-deficient models, where thioredoxin system upregulation maintained redox balance despite GSR loss .

What role do GSR antibodies play in investigating tissue-specific antioxidant defense mechanisms?

GSR antibodies are instrumental in elucidating tissue-specific antioxidant defense mechanisms through several specialized applications:

Multiplex immunohistochemistry combining GSR antibodies with other antioxidant enzymes (SOD, catalase, GPX) allows for comprehensive tissue-specific mapping of antioxidant defense systems. Using sequential staining protocols with GSR antibody dilutions of 1:200-1:800 , researchers can visualize the relative expression and co-localization patterns across different tissue regions. This approach revealed that in cochlear cells, the glutathione system represents a major defense mechanism for protection .

Tissue microarray analysis with GSR antibodies across multiple organs and disease states can identify tissue-specific variations in GSR expression patterns. The Human Protein Atlas project utilizes such approaches with antibodies like HPA001538 to systematically map protein expression across 44 normal human tissues and 20 common cancer types. These datasets reveal that GSR expression varies significantly between tissues, reflecting different antioxidant requirements.

Laser capture microdissection combined with immunohistochemistry and proteomics allows for region-specific analysis of GSR expression within heterogeneous tissues. This technique has been particularly valuable in neural tissues and cochlea, where specific cellular populations (e.g., hair cells, stria vascularis) show differential antioxidant enzyme activities in response to stressors like noise exposure .

Comparative studies across species using cross-reactive GSR antibodies (validated for human, mouse, and rat samples ) can identify evolutionary conservation or divergence in tissue-specific antioxidant mechanisms. This approach has revealed that while basic GSR function is conserved, regulatory mechanisms and interactions with other antioxidant systems vary between species and tissues, informing translational research approaches.

How can GSR antibodies facilitate research on disease models related to oxidative stress?

GSR antibodies offer valuable tools for investigating disease models related to oxidative stress through several sophisticated approaches:

Longitudinal analysis of GSR expression and localization during disease progression can be achieved through time-course immunohistochemistry studies using GSR antibodies at 1:200-1:800 dilutions . In neurodegenerative models, this approach has revealed dynamic changes in GSR expression that precede clinical symptoms, potentially identifying early biomarkers. Correlating these expression patterns with oxidative damage markers and functional outcomes provides mechanistic insights into disease pathogenesis.

Therapeutic intervention assessment using GSR antibodies can evaluate how pharmacological or genetic treatments affect antioxidant defense systems. Western blot analysis with GSR antibodies at 1:1000-1:4000 dilutions can quantify changes in GSR expression following treatment, while activity assays paired with immunoprecipitation can assess functional impacts. This combined approach distinguishes between increased expression versus enhanced activity of existing enzyme.

Cross-platform validation employing both GSR antibody-based detection and functional GSR activity assays provides comprehensive assessment of redox defense status. Studies in Meniere's disease patients demonstrated decreased GSH/GSSG ratios in plasma and lymphocytes , which could be correlated with tissue-specific GSR expression patterns detected by immunohistochemistry to establish relationships between systemic and local antioxidant capacity.

Single-cell analysis techniques using GSR antibodies for immunofluorescence (1:50-1:500 dilutions) combined with flow cytometry can identify cellular subpopulations with differential GSR expression within heterogeneous tissues or disease lesions. This approach has revealed that not all cells within a tissue respond uniformly to oxidative challenges, with implications for understanding disease susceptibility and progression at the cellular level.

What are emerging applications for GSR antibodies in cutting-edge research technologies?

GSR antibodies are finding new applications in several cutting-edge research technologies:

Mass cytometry (CyTOF) coupled with metal-conjugated GSR antibodies enables high-dimensional analysis of antioxidant defense systems at the single-cell level. This technology allows simultaneous detection of GSR alongside dozens of other markers, creating comprehensive profiles of cellular redox status across heterogeneous populations. Such approaches could reveal previously unrecognized cell subpopulations with distinct antioxidant capabilities relevant to disease susceptibility.

CRISPR screens targeting redox regulation pathways can be validated using GSR antibodies for phenotypic confirmation. Western blot analysis with 1:1000-1:4000 antibody dilutions provides efficient validation of knockout efficiency, while immunofluorescence at 1:50-1:500 dilutions can assess subcellular localization changes in interacting partners. This combined approach strengthens the interpretation of genetic screen results by connecting genomic alterations to protein-level consequences.

Spatial transcriptomics paired with GSR immunohistochemistry creates powerful datasets linking protein expression with local transcriptional landscapes. By applying GSR antibodies at 1:200-1:800 dilutions to serial sections used for spatial transcriptomics, researchers can correlate GSR protein distribution with gene expression patterns of related antioxidant pathways, revealing regulatory networks that coordinate redox defense systems across tissue microenvironments.

Microfluidic tissue models ("organs-on-chips") combined with real-time imaging of GSR dynamics using fluorescently-labeled antibodies or reporter systems can track antioxidant responses to controlled oxidative challenges. This emerging approach allows for dynamic visualization of GSR regulation under precisely defined conditions, offering insights into the temporal aspects of redox defense activation that cannot be captured in fixed tissue analyses.

How might GSR antibody applications evolve in studying the intersection of redox biology with other cellular pathways?

The application of GSR antibodies in studying the intersection of redox biology with other cellular pathways is evolving in several promising directions:

Metabolic reprogramming studies are increasingly incorporating GSR antibodies to investigate links between redox balance and cellular metabolism. Recent research has demonstrated that glutaredoxins, working alongside optimal ROS levels, can activate AMPK through S-glutathionylation to improve glucose metabolism in type 2 diabetes . GSR antibodies enable researchers to track how changes in glutathione reduction capacity influence metabolic enzyme activity and energy production pathways through post-translational modifications.

Cell fate determination research is utilizing GSR antibodies to explore how redox states influence differentiation, senescence, and programmed death pathways. Studies have shown that antioxidant defense in quiescent cells determines selectivity of electron transport chain inhibition-induced cell death , with GSR playing a key role in maintaining GSH pools that influence these decisions. Combining GSR immunofluorescence with markers of cell fate provides spatial context for understanding how local redox environments shape cellular decisions.

Immune function studies are applying GSR antibodies to investigate redox regulation of inflammatory responses. Research has demonstrated that Gsr deficiency results in defects in host defense against bacterial infection , highlighting critical roles for glutathione in immune cell function. Multiplex immunohistochemistry combining GSR with immune cell markers and activation status indicators can map how redox capacity correlates with immune competence across tissue microenvironments.

Epigenetic regulation research is beginning to incorporate GSR antibodies to explore connections between redox states and chromatin modifications. Emerging evidence suggests that GSSG/GSH ratios can influence histone-modifying enzyme activity and DNA methylation patterns. ChIP-sequencing paired with GSR expression analysis could reveal how alterations in glutathione homeostasis reshape the epigenetic landscape, potentially explaining long-term consequences of transient oxidative stress episodes.

What are the most reliable controls and validation strategies for GSR antibody-based experiments?

For maximizing reliability in GSR antibody-based experiments, implement these comprehensive validation strategies:

Positive and negative tissue/cell controls should be selected based on validated expression patterns. Use Jurkat cells, HeLa cells, mouse lung tissue, or human placenta tissue as positive controls for Western blot applications . For negative controls, consider using samples with confirmed GSR knockdown or tissues with naturally low GSR expression. When possible, GSR knockout models provide the gold standard negative control, though researchers should be aware that complete knockouts may trigger compensatory upregulation of related pathways like the thioredoxin system .

Multiple antibody validation requires testing at least two independent antibodies targeting different GSR epitopes. Commercial antibodies like ab137513 (targeting amino acids 150-450) and 18257-1-AP can be compared for consistent detection patterns. Observed molecular weights should align with predicted values (56 kDa for monomers, approximately 110 kDa for dimers) . Discrepancies between antibodies should be investigated through additional validation steps.

Functional correlation studies should link antibody-detected GSR levels with enzymatic activity. This approach is particularly important given findings that GSR-deficient mice showed significant decreases in GSR activity and GSH/GSSG ratios in the cytosol of inner ears . Establish a standard curve relating band intensity or staining intensity to quantitative activity measurements to strengthen the biological relevance of antibody-based detection.

Reproducibility verification requires detailed documentation of protocols including antibody dilutions (WB: 1:1000-1:4000, IHC: 1:200-1:800, IF: 1:50-1:500) , incubation conditions, and detection methods. Include quantifiable parameters (band intensity, positive cell percentages) with appropriate statistical analysis to enable robust comparison across experiments and laboratories.

What are key considerations for designing longitudinal studies involving GSR antibody detection?

Designing effective longitudinal studies involving GSR antibody detection requires careful planning around several key considerations:

Sample collection standardization is crucial for temporal comparisons. Establish strict protocols for tissue collection, processing, and storage to minimize technical variability. For biobanked specimens, document preservation methods and storage duration, as these factors can affect epitope integrity and detection sensitivity . When working with human samples, standardize collection timing relative to treatments or disease stages, as GSR expression and activity can fluctuate with acute oxidative challenges.

Batch effects mitigation requires processing temporal samples simultaneously when possible. If processing must occur in batches, include reference standards and internal controls across all batches to enable normalization. For immunohistochemistry applications (using 1:200-1:800 antibody dilutions) , stain control and experimental samples on the same slides or in the same batch to minimize technical variability in staining intensity. Include calibration standards in every Western blot to enable accurate cross-blot comparisons.

Multi-parameter analysis strengthens longitudinal studies by correlating GSR with functional outcomes. Beyond antibody-based detection, measure GSH/GSSG ratios, oxidative damage markers, and functionally related enzymes (thioredoxin, thioredoxin reductase) whose activities may increase to compensate for GSR deficiency . This comprehensive approach provides mechanistic insights into how changing GSR expression influences cellular redox homeostasis over time.

Statistical power calculations for longitudinal designs must account for expected biological variability in GSR expression, potential subject attrition, and temporal resolution requirements. Power analyses should guide sample size determination, with consideration for subgroup analyses that may reveal population-specific patterns. Minority subjects may show different participation rates in longitudinal biospecimen studies , potentially affecting population representation in GSR research over time.