GGT1 Antibody

What is GGT1 and why are antibodies against it important for research?

GGT1 (gamma-glutamyltransferase 1) is a membrane-bound enzyme that plays a crucial role in glutathione metabolism by acting as a glutathionase that transfers the glutamyl moiety from glutathione to various amino acids and dipeptide acceptors . This enzymatic activity is essential for:

• Maintaining cellular redox balance

• Detoxifying harmful compounds

• Supporting cellular defense mechanisms
  GGT1 primarily resides on the outer cell membrane surface and is expressed across mammalian tissues, with particularly high expression in the liver, kidney, epididymis, stomach, and urinary bladder . Its importance in research stems from:

• Its role as an early biomarker for hepatocellular carcinoma and liver damage

• Tissue-specific glycosylation patterns that change in disease states

• Significant heterogeneity in human serum and tissues due to different glycosylation rather than gene product variations
  Antibodies against GGT1 enable precise detection of these variations in expression and post-translational modifications, making them invaluable tools for studying both normal physiology and pathological conditions.

What are the main types of GGT1 antibodies available for research purposes?

Researchers have several options when selecting GGT1 antibodies, each with distinct characteristics:
By host species and type:

• Mouse monoclonal: e.g., clone 1F9 (IgG2a isotype) and E-5 (IgG1 kappa light chain)

• Rabbit monoclonal: e.g., clone EPR5288

• Rabbit polyclonal antibodies

• Recombinant antibodies
  By reactivity:

• Human-specific

• Mouse-specific

• Multi-species (human/mouse/rat)
  By target epitope:

• N-terminal targeting antibodies

• C-terminal targeting antibodies (e.g., GGT129 polyclonal)

• Specific domain recognition (e.g., amino acids 381-471)
  By conjugation:

• Unconjugated forms

• Conjugated variants including:

  • Agarose

  • Horseradish peroxidase (HRP)

  • Phycoerythrin (PE)

  • Fluorescein isothiocyanate (FITC)

  • Multiple Alexa Fluor® options
    The selection depends on your specific application, with monoclonal antibodies offering higher specificity and polyclonal antibodies providing better sensitivity for detecting low-abundance targets.

What are the validated applications for GGT1 antibodies and their recommended dilutions?

GGT1 antibodies have been validated for multiple applications with specific dilution ranges:

• 7.5% SDS-PAGE for WB with 30 μg of various cell lysates (Neuro2A, GL261, C8D30, NIH-3T3, Raw264.7, C2C12)

• Positive WB detection in human kidney tissue

• Positive IHC detection in human intrahepatic cholangiocarcinoma tissue
  Important note: Always perform proper validation with positive and negative controls for your specific experimental system. For IHC applications, antigen retrieval methods significantly impact results, with some antibodies performing optimally with TE buffer pH 9.0 while others require citrate buffer pH 6.0 .

How should researchers validate GGT1 antibodies for their specific experimental systems?

Comprehensive antibody validation should follow these methodological steps:
1. Initial specificity assessment:

• Analyze manufacturer validation data (Western blots, IHC images)

• Check literature for previous validation in your model system

• Verify the immunogen sequence used for antibody production to assess potential cross-reactivity
  2. Experimental validation protocol:

• Positive controls: Use tissues known to express high levels of GGT1 (liver, kidney, epididymis)

• Negative controls: Include samples from knockdown/knockout models or tissues with minimal GGT1 expression

• Antibody controls: Include secondary antibody-only controls to detect non-specific binding

• Epitope blocking: Pre-incubate antibody with immunizing peptide to confirm specificity
  3. Application-specific validation:

• For Western blot: Confirm band size (expected: ~65-70 kDa for full protein; light chain ~24 kDa) and compare multiple antibodies

• For IHC: Compare staining patterns with published data and RNA expression databases

• For IF/ICC: Verify subcellular localization matches expected membrane distribution
  4. Cross-application validation:
  Confirm consistency between techniques (e.g., does WB detection match IHC localization?)
  Statistical considerations:

• Run technical triplicates

• Use appropriate statistical tests for quantitative analyses

• Include biological replicates to account for sample variation
  The antibody-lectin sandwich array (ALSA) technique described in search result offers an innovative approach to validation by confirming both antibody binding and glycosylation patterns simultaneously, providing multi-dimensional verification of specificity.

What are the key methodological considerations for using GGT1 antibodies in tissue immunohistochemistry?

Successful immunohistochemistry with GGT1 antibodies requires careful attention to several methodological details:
Sample preparation:

• Use freshly prepared 10% neutral buffered formalin for fixation (12-24 hours optimal)

• Process tissues within standardized schedules to avoid over-fixation

• Use positively charged slides to prevent tissue detachment
  Antigen retrieval (critical for GGT1):

• Method selection: GGT1 antibodies typically require heat-induced epitope retrieval (HIER)

• Buffer options:

  • Sodium citrate buffer (pH 6.0) - standard approach

  • TE buffer (pH 9.0) - often yields better results with recombinant antibodies

• Duration: 15-20 minutes at controlled temperature (95-100°C)
  Antibody optimization:

• Titration: Test multiple dilutions (1:50 to 1:2000 range)

• Incubation conditions: 1 hour at room temperature or overnight at 4°C

• Detection systems: HRP-polymer systems typically provide better signal-to-noise ratio than avidin-biotin methods
  Controls and validation:

• Include positive control tissues (human liver or kidney sections)

• Use isotype controls to assess background staining

• Consider dual staining with alternative GGT1 antibodies targeting different epitopes
  Special considerations:

• GGT1 shows significant glycosylation heterogeneity that varies between tissues

• Normal liver and kidney GGT1 have distinct glycosylation patterns that affect epitope accessibility

• Tumors may exhibit altered glycosylation patterns that change antibody recognition
  For semi-quantitative analysis, establish a scoring system based on staining intensity and percentage of positive cells, with at least two independent pathologists scoring blinded samples.

How can researchers troubleshoot common issues with GGT1 antibody applications?

• First validate antibody with known positive control (e.g., human kidney tissue for WB )

• Implement changes one at a time to identify problematic variable

• Document all optimization steps and results

• Consider using alternative antibody clones/vendors if problems persist
  For glycosylation heterogeneity issues specific to GGT1, specialized techniques like lectin affinity enrichment or glycosidase treatment prior to Western blotting may help resolve complex banding patterns .

How can antibody-lectin sandwich arrays (ALSA) be used to study GGT1 glycosylation patterns in disease models?

The antibody-lectin sandwich array (ALSA) platform represents an advanced microanalytical technique that enables researchers to characterize the N-glycan content of GGT1 in complex biological samples using minimal input material:
Methodological principles:

• GGT1-specific antibody is immobilized and chemically derivatized to prevent recognition by lectins

• The antibody captures GGT1 from complex samples (e.g., tissue extracts)

• Different lectins with specific carbohydrate-binding preferences probe the glycan structures on the captured GGT1

• The distinctive binding patterns reveal glycosylation differences
  Implementation protocol:

• Sample preparation: Prepare detergent-extracted membrane proteins from tissues or cells

• Antibody immobilization: Use a polyclonal antibody (e.g., GGT129) targeting a non-glycosylated epitope

• Capture verification: Confirm equivalent capture efficiency across samples

• Lectin probing: Apply a panel of lectins with known glycan-binding specificities

• Detection: Use labeled secondary reagents to quantify lectin binding
  Advantages for GGT1 research:

• Requires sub-microgram quantities of total protein compared to traditional glycoprotein analysis methods

• Can detect distinct glycosylation patterns between normal human liver GGT1, normal human kidney GGT1, and recombinant GGT1

• Enables identification of tumor-specific glycosylation changes that may serve as biomarkers

• Can implement specialized lectins like microvirin (MVN) that specifically recognize high-mannose-type N-glycans characteristic of tumor-derived GGT1
  Detection sensitivity:
  The ALSA platform achieves a detection limit of approximately 0.3 nanograms for GGT1, making it suitable for analysis of clinical specimens with limited material availability .
  This technique provides researchers with a powerful tool for high-throughput screening of disease-related changes in GGT1 glycosylation patterns, potentially supporting diagnostic development and therapeutic monitoring strategies.

What are the methodological approaches for studying the different isoforms and post-translational modifications of GGT1?

GGT1 exists in multiple isoforms with complex post-translational modifications that require specialized methodological approaches:
Isoform characterization strategies:

• Include tissue panels from normal and disease states

• Compare liver, kidney, and tumor tissues which display distinct glycosylation patterns

• Normalize samples by GGT1 enzymatic activity (for functional studies) or total protein content (for expression studies)
  The heterogeneity of GGT1 in human serum and tissues is primarily due to different glycosylation patterns rather than gene product variations , making glycoanalysis particularly important for understanding its role in health and disease.

How can researchers effectively use GGT1 antibodies in multiplex immunoassays and co-localization studies?

Setting up robust multiplex and co-localization studies with GGT1 antibodies requires careful methodological planning:
Multiplex immunoassay design:

• Antibody selection criteria:

  • Choose antibodies from different host species to avoid cross-reactivity

  • Verify each antibody works independently before multiplexing

  • Consider using directly conjugated primary antibodies when possible

• Panel design strategies:

  • For flow cytometry: Combine GGT1 (CD224) with other membrane markers

  • For IHC/IF: Pair GGT1 with cell-type specific markers to identify expressing populations

  • Spectral considerations: Ensure fluorophores have sufficient spectral separation

• Optimized protocol development:

  • Test sequential vs. simultaneous antibody incubation

  • Adjust concentrations of each antibody independently

  • Include robust controls for each marker
    Co-localization experimental approach:

• Sample preparation optimization:

  • Use thin sections (5μm or less) for tissue imaging

  • For cells, ensure proper fixation preserves membrane structures where GGT1 localizes

• Technical considerations:

  • Confocal parameters: Use appropriate pinhole settings to minimize bleed-through

  • Image acquisition: Capture sequential channels when spectral overlap exists

  • Resolution: Super-resolution techniques may better resolve membrane localization

• Analysis methods:

  • Calculate Pearson's correlation coefficient for quantitative co-localization

  • Use line-scan analysis across cellular structures

  • Apply deconvolution to improve spatial resolution
    Practical example protocol for GGT1/marker co-localization:

• Fix tissues/cells with 4% paraformaldehyde (10-15 minutes)

• Permeabilize with 0.1% Triton X-100 if necessary

• Block with 5% normal serum (1 hour)

• Incubate with mouse anti-GGT1 [1F9] (1:100) and rabbit anti-marker antibody (optimized dilution)

• Wash 3x with PBS

• Apply species-specific secondary antibodies with distinct fluorophores

• Counterstain nuclei with DAPI

• Mount with anti-fade medium

• Image with confocal microscopy

• Analyze co-localization with appropriate software (ImageJ with JACoP plugin)
  This approach is particularly valuable for studying GGT1's relationship with other membrane proteins in specific cellular compartments and tissue microenvironments.

What experimental strategies can identify and validate GGT1 as a biomarker in clinical specimens?

Developing GGT1 as a clinically relevant biomarker requires a comprehensive experimental approach that leverages its unique tissue-specific post-translational modifications:
Biomarker discovery phase:

• Tissue profiling strategy:

  • Compare GGT1 expression across normal vs. disease tissues using IHC

  • Analyze glycosylation patterns using the ALSA platform

  • Screen a discovery cohort with sufficient statistical power

  • Capture clinicopathological variables for correlation analysis

• Detection method development:

  • Tissue-based: Optimize IHC protocols with antigen retrieval parameters

  • Serum-based: Develop specific immunoassays for circulating GGT1

  • Activity-based: Correlate enzyme activity with protein levels
    Validation methodology:

• Analytical validation:

  • Determine assay sensitivity and specificity

  • Establish reproducibility (intra- and inter-assay CV <15%)

  • Define standard curves and dynamic range

  • Set quality control parameters

• Clinical validation protocol:

  • Use independent patient cohorts

  • Include early-stage and late-stage disease

  • Compare against current gold standard biomarkers

  • Calculate ROC curves with AUC values

  • Determine positive/negative predictive values
    Implementation considerations:

• Sample requirements:

  • Define optimal collection and processing procedures

  • Establish stability parameters under various storage conditions

  • Determine minimal sample input requirements (ALSA platform requires only sub-microgram quantities)

• Data integration approach:

  • Develop multivariate models combining GGT1 with other biomarkers

  • Incorporate clinicopathological variables

  • Use machine learning algorithms for pattern recognition
    Specific GGT1 applications:

• Liver diseases: GGT1 enters the bloodstream following liver damage and serves as an early biomarker for hepatocellular carcinoma

• Tumor detection: High-mannose-type N-glycans on GGT1 appear unique to malignant kidney and liver tissues and can be detected using the microvirin lectin

• Treatment monitoring: Sequential sampling to track changes in GGT1 levels or glycosylation patterns during therapy The unique glycosylation heterogeneity of GGT1 offers potential for developing highly specific biomarkers that can distinguish between different pathological conditions beyond what is possible with activity measurements alone.