GSS Antibody, HRP conjugated

What is GSS Antibody, HRP conjugated and what is its molecular structure?

GSS (Glutathione synthetase) is a 474 amino acid protein encoded by a gene located on human chromosome 20q11.2. The GSS antibody conjugated with HRP (Horseradish Peroxidase) is specifically designed to detect this protein in various experimental applications. The target protein GSS consists of three loops projecting from an antiparallel β-sheet, a parallel β-sheet, and a lid of anti-parallel sheets that provide access to the ATP-binding site . The crystal structure indicates that GSS belongs to the ATP-GRASP superfamily, despite Southern blot and gene analysis suggesting it may be the only member of a unique family .

The HRP-conjugated antibody is typically a polyclonal IgG raised in rabbit hosts using KLH-conjugated synthetic peptides derived from human Glutathione Synthetase as immunogens . The immunogen range generally encompasses amino acids 81-160 of the 474 amino acid sequence, which provides optimal epitope recognition .

What are the primary research applications for GSS Antibody, HRP conjugated?

GSS Antibody, HRP conjugated is suitable for multiple experimental applications, including:

• Western Blotting (WB): For detecting denatured GSS protein in cell or tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of GSS in solution

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): For visualizing GSS localization in fixed tissue sections

The antibody demonstrates confirmed reactivity with human, mouse, and rat samples, with predicted cross-reactivity extending to samples from dog, cow, sheep, pig, and horse sources based on sequence homology . This broad species reactivity makes it valuable for comparative studies across multiple model organisms.

How does GSS Antibody differ from GST Antibody, despite similar nomenclature?

Despite the similar acronyms, GSS (Glutathione Synthetase) and GST (Glutathione S-transferase) are distinct proteins with different functions in glutathione metabolism:

Understanding this distinction is crucial when selecting antibodies for experiments, as anti-GST antibodies are frequently used to detect GST-tagged recombinant proteins rather than endogenous GST .

What are the optimal conditions for using GSS Antibody, HRP conjugated in Western blotting?

For optimal Western blotting results with GSS Antibody, HRP conjugated, researchers should implement the following methodological approach:

• Sample preparation: Extract proteins using a buffer containing protease inhibitors to prevent degradation of GSS (52 kDa)

• Gel electrophoresis: Use 10-12% SDS-PAGE gels for optimal separation

• Transfer: Implement semi-dry or wet transfer at 100V for 60-90 minutes

• Blocking: Block membranes with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation: Dilute GSS Antibody, HRP conjugated to 1:1000-1:2000 in blocking buffer and incubate overnight at 4°C

• Detection: Since the antibody is HRP-conjugated, secondary antibody incubation is not required. Proceed directly to chemiluminescent detection

The HRP conjugation provides direct enzymatic detection capability, eliminating the need for secondary antibodies and thereby reducing background and cross-reactivity issues that can arise in multi-step detection protocols .

How should researchers optimize GSS Antibody, HRP conjugated for ELISA applications?

When using GSS Antibody, HRP conjugated in ELISA, consider these methodological optimizations:

• Coating concentration: For indirect ELISA, coat plates with 1-10 μg/ml of target antigen

• Blocking: Use 1% BSA in PBS to minimize background

• Antibody dilution: Begin with 1:1000 dilution of HRP-conjugated GSS antibody and perform titration to determine optimal concentration

• Incubation time: Incubate for 1-2 hours at room temperature or overnight at 4°C for maximum sensitivity

• Substrate selection: For HRP detection, TMB (3,3',5,5'-Tetramethylbenzidine) or OPD (o-phenylenediamine) are recommended substrates

• Signal measurement: Measure absorbance at 450 nm for TMB or 492 nm for OPD

When compared to conventional secondary antibody detection systems, directly HRP-conjugated antibodies can provide enhanced signal with reduced background, particularly at medium to high concentrations of target antigen . For detecting low abundance GSS targets, signal amplification techniques such as TSA (Tyramide Signal Amplification) may be incorporated.

What troubleshooting approaches should be implemented for non-specific binding issues?

Non-specific binding is a common challenge when using HRP-conjugated antibodies. Researchers can implement these methodological solutions:

• Increase blocking agent concentration: Try 5% BSA or 5% non-fat milk in TBST

• Add 0.1-0.5% Tween-20 to washing and antibody dilution buffers

• Pre-absorb the antibody with proteins from non-target species

• Include competitive inhibitors: Add 1-5% normal serum from the same species as the samples

• Optimize antibody concentration: Titrate to determine the minimum effective concentration

• Increase washing frequency and duration between steps

• Incorporate negative controls: Include samples known to be negative for GSS to evaluate background levels

For particularly stubborn background issues, consider preparing a blocking solution containing both protein blockers (BSA or milk) and non-ionic detergents (Tween-20 or Triton X-100) to disrupt both protein-protein and hydrophobic interactions .

How can GSS Antibody, HRP conjugated be utilized in multiplex immunodetection systems?

For advanced multiplex detection systems involving GSS Antibody, HRP conjugated, researchers should consider:

• Sequential multiplex protocol:

  • First round: Apply GSS Antibody, HRP conjugated and develop with a precipitating substrate

  • Strip the membrane using a mild stripping buffer (e.g., glycine-SDS buffer, pH 2.2)

  • Verify complete stripping using detection reagent

  • Re-block and apply the second antibody targeting a different protein

• Spectral multiplexing:

  • Use GSS Antibody, HRP conjugated with a specific fluorescent tyramide substrate

  • Quench the peroxidase activity using H₂O₂ or sodium azide

  • Apply the next HRP-conjugated antibody with a different fluorescent tyramide

  • Repeat for additional targets

  • Image using appropriate filters for each fluorophore

The species-independent binding capability of properly designed secondary detection systems makes them versatile for multiple applications regardless of the species origin of primary antibodies . When designing multiplex experiments, consider the subcellular localization of GSS (primarily cytoplasmic) to avoid potential signal overlap with other targets .

What approaches can enhance signal detection for low-abundance GSS protein?

For detecting low-abundance GSS protein, researchers can implement these signal enhancement strategies:

• Tyramide Signal Amplification (TSA):

  • The HRP conjugate catalyzes the deposition of multiple tyramide molecules

  • This can increase sensitivity by 10-100 fold compared to conventional detection

  • Particularly effective for IHC-P applications with limited antigen availability

• Optimized sample preparation:

  • Implement protein concentration techniques (e.g., TCA precipitation)

  • Use phosphatase inhibitors to preserve post-translational modifications

  • Consider subcellular fractionation to enrich for cytoplasmic proteins

• Enhanced chemiluminescent detection:

  • Use supersensitive ECL substrates with extended signal duration

  • Implement signal accumulation through multiple exposures

  • Consider digital imaging systems with cooling capabilities to reduce noise

When comparing HRP-conjugated primary antibodies to conventional primary-secondary systems, studies have shown comparable limits of detection (20-30 pM range) but enhanced signal intensity at medium to high concentrations . This makes HRP-conjugated GSS antibodies particularly valuable for quantitative applications requiring robust signal.

How does the temperature stability of GSS Antibody, HRP conjugated affect experimental design?

Temperature stability is a critical consideration when working with HRP-conjugated antibodies:

• Storage conditions:

  • Store at -20°C to maintain activity

  • Aliquot into multiple vials to avoid repeated freeze-thaw cycles

  • The storage buffer (typically containing 0.01M TBS pH 7.4, 1% BSA, 0.03% Proclin300, and 50% Glycerol) stabilizes both antibody and HRP enzyme

• Temperature effects on experimental protocols:

  • HRP activity is temperature-dependent, with optimal activity at 25-30°C

  • Prolonged exposure to temperatures above 37°C can permanently damage HRP activity

  • Reaction kinetics approximately double with every 10°C increase in temperature

• Methodological considerations:

  • Pre-equilibrate all reagents to room temperature before use

  • For longer incubations (>2 hours), conduct at 4°C to preserve antibody-antigen binding without compromising HRP stability

  • When extending reaction times for enhanced sensitivity, maintain consistent temperature to ensure reproducible results

Temperature fluctuations can significantly impact data reproducibility, particularly in quantitative applications. Researchers should implement temperature monitoring and control measures throughout their experimental workflow .

What are the optimal approaches for quantitative analysis of GSS expression using HRP-conjugated antibodies?

For rigorous quantitative analysis of GSS expression using HRP-conjugated antibodies, implement these methodological approaches:

• Standard curve generation:

  • Create a standard curve using recombinant GSS protein at known concentrations

  • Plot absorbance or chemiluminescence against concentration

  • Use semi-logarithmic scale for wide dynamic range applications

• Signal normalization strategies:

  • Normalize to total protein concentration (determined by Bradford or BCA assay)

  • Use housekeeping proteins (β-actin, GAPDH) as loading controls

  • Implement direct total protein normalization through stain-free gels or membrane staining

• Image acquisition and analysis:

  • Capture multiple exposures to ensure linearity of signal

  • Use dedicated analysis software with background subtraction capabilities

  • Perform densitometric analysis of bands using integrated density values

• Statistical analysis:

  • Apply appropriate statistical tests based on experimental design (t-test, ANOVA)

  • Calculate coefficient of variation (CV) between replicates (<15% is acceptable)

  • Report results with error bars representing standard deviation or standard error

The linear response range for HRP-conjugated antibodies typically spans approximately 40 pM to 5 nM of target, with comparable limits of detection to conventional secondary antibody systems . For most accurate quantitation, design experiments to fall within this linear range.

How should researchers interpret cross-reactivity data for GSS Antibody, HRP conjugated?

Interpreting cross-reactivity data requires careful methodological consideration:

• Predicted vs. confirmed reactivity:

  • GSS Antibody has confirmed reactivity with human, mouse, and rat samples

  • Predicted reactivity extends to dog, cow, sheep, pig, and horse based on sequence homology

  • When working with predicted-reactive species, validation experiments are essential

• Validation approaches:

  • Positive control: Use samples with known GSS expression

  • Negative control: Use GSS-knockout or knockdown samples

  • Peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

  • Multiple antibody comparison: Use antibodies targeting different GSS epitopes

• Cross-reactivity interpretation framework:

  • Strong signal with expected molecular weight: Likely specific binding

  • Multiple bands: Potential splice variants, degradation products, or non-specific binding

  • Unexpected molecular weight band only: Likely non-specific binding

Researchers should note that the immunogen range for GSS Antibody (amino acids 81-160/474) may impact cross-reactivity patterns based on the conservation of this region across species . Sequence alignment analysis prior to experimental design can help predict potential cross-reactivity issues.

What considerations are important when comparing data across different detection systems using GSS Antibody, HRP conjugated?

When comparing data from different detection systems:

• Detection system calibration:

  • Create a common reference sample to run across all systems

  • Establish conversion factors between different readout units

  • Document system-specific parameters (exposure time, gain settings, etc.)

• System-specific characteristics:

  • Colorimetric: Limited dynamic range but stable signal

  • Chemiluminescent: Wider dynamic range but time-dependent signal decay

  • Fluorescent: Good dynamic range with potential for photobleaching

• Data normalization strategies:

  • Use internal controls consistently across all systems

  • Apply system-specific background correction

  • Consider using ratio metrics rather than absolute values

• Methodological documentation:

  • Record detailed protocols including substrate type, incubation time, and detection parameters

  • Note lot numbers of GSS Antibody, HRP conjugated used across experiments

  • Maintain consistent antibody concentration across detection systems

What are the optimal storage conditions for maintaining GSS Antibody, HRP conjugated activity?

Proper storage is critical for maintaining antibody and HRP activity:

• Temperature requirements:

  • Store at -20°C for long-term preservation

  • Avoid storage at 4°C for more than 1-2 weeks

  • Never store at room temperature

• Aliquoting recommendations:

  • Divide into single-use aliquots immediately upon receipt

  • Use sterile tubes with secure seals

  • Indicate concentration and date on each aliquot

  • Avoid more than 5 freeze-thaw cycles

• Buffer considerations:

  • The storage buffer (0.01M TBS pH 7.4 with 1% BSA, 0.03% Proclin300, and 50% Glycerol) is optimized for both antibody and HRP stability

  • Do not dilute the stock solution until ready for use

  • If dilution is necessary, prepare fresh working solutions daily

Proper storage can maintain antibody activity for up to 12 months, while improper handling can significantly reduce shelf life and experimental reproducibility.

How should dilution factors be determined for different experimental applications?

Determining optimal dilution factors requires methodical titration:

• Initial dilution range recommendations:

  • Western blotting: 1:500 to 1:5000

  • ELISA: 1:1000 to 1:10,000

  • IHC-P: 1:100 to 1:1000

• Titration methodology:

  • Prepare a minimum of 5 serial dilutions covering the recommended range

  • Use consistent sample and protocol conditions

  • Analyze signal-to-noise ratio at each dilution

  • Select the dilution that provides maximum specific signal with minimal background

• Application-specific considerations:

  • Higher antibody concentrations are typically required for IHC-P due to potential epitope masking during fixation

  • Lower concentrations often suffice for ELISA due to direct antigen binding

  • Western blotting often requires intermediate concentrations

The concentration of GSS Antibody, HRP conjugated is typically 1μg/μl , facilitating precise dilution calculations for experimental applications.

What quality control metrics should researchers implement when working with GSS Antibody, HRP conjugated?

Implementing robust quality control ensures experimental reliability:

• Initial validation tests:

  • Positive control: Test with samples known to express GSS

  • Negative control: Test with samples known to lack GSS expression

  • Dilution linearity: Confirm signal proportionality with dilution

• Ongoing quality monitoring:

  • Include consistent positive control in each experiment

  • Track signal intensity across experiments

  • Monitor background levels in negative controls

  • Document lot-to-lot variation when receiving new antibody stock

• Troubleshooting indicators:

  • Sudden loss of signal: Potential antibody degradation or HRP inactivation

  • Increasing background: Possible contamination or non-specific binding

  • Variable signal between replicates: Inconsistent technique or reagent stability issues

The HRP conjugation provides signal amplification capabilities with approximately 3 HRP molecules per antibody molecule, which should yield consistent signal enhancement compared to conventional two-step detection systems .

How can researchers troubleshoot weak or absent signal when using GSS Antibody, HRP conjugated?

When encountering weak or absent signal, methodically address potential issues:

• Antibody-related factors:

  • Check storage conditions and freeze-thaw history

  • Verify HRP activity using direct substrate test

  • Confirm antibody concentration and dilution calculation

  • Consider using a new lot or alternative antibody targeting different epitope

• Sample-related factors:

  • Verify protein extraction efficiency

  • Check for protease activity in samples

  • Assess protein loading amount

  • Consider epitope masking due to protein modifications or complex formation

• Protocol optimizations:

  • Increase antibody concentration or incubation time

  • Optimize antigen retrieval for IHC-P applications

  • Try alternative blocking agents

  • Implement signal amplification techniques like TSA

• Detection system checks:

  • Verify substrate freshness and activity

  • Increase substrate incubation time

  • Try alternative, more sensitive detection substrates

  • Check imaging system settings and sensitivity

For particularly low abundance targets, adjusting to the linear detection range (approximately 40 pM to 5 nM) can significantly improve signal quality .

How can GSS Antibody, HRP conjugated be used in mechanistic studies of glutathione synthesis pathways?

GSS Antibody, HRP conjugated offers valuable approaches for mechanistic studies:

• Pathway perturbation analysis:

  • Track GSS protein levels following pathway inhibition

  • Monitor compensatory responses to glutathione depletion

  • Assess GSS expression during oxidative stress responses

• Co-localization studies:

  • Use GSS Antibody, HRP conjugated with TSA-based fluorescent detection

  • Implement multiplexed imaging with other pathway components

  • Analyze subcellular distribution in response to cellular stressors

• Protein-protein interaction studies:

  • Combine with proximity ligation assays to detect GSS interactions

  • Assess complex formation with pathway partners

  • Evaluate changes in interaction patterns under different conditions

• Disease mechanism investigation:

  • Compare GSS expression in normal versus pathological tissues

  • Correlate expression with disease biomarkers

  • Evaluate therapeutic modulation of the glutathione synthesis pathway

Understanding GSS structure, including its three loops projecting from antiparallel β-sheets and the ATP-binding site, provides insights into its functional regulation within the glutathione synthesis pathway .

What approaches can enable simultaneous detection of multiple glutathione pathway components?

For comprehensive glutathione pathway analysis:

• Sequential multiplex Western blotting:

  • First detection: Use GSS Antibody, HRP conjugated

  • Strip membrane: Use mild stripping buffer

  • Second detection: Probe for glutamate-cysteine ligase (GCL)

  • Additional cycles: Detect glutathione peroxidase (GPx) and other pathway components

• Multiplex immunohistochemistry:

  • First round: GSS Antibody, HRP conjugated with TSA-Fluorophore 1

  • Quenching: Use H₂O₂ to inactivate HRP

  • Subsequent rounds: Additional antibodies with different fluorophores

  • Analysis: Multi-channel confocal microscopy with spectral unmixing

• Bead-based multiplex ELISA:

  • Conjugate different pathway antibodies to spectrally distinct beads

  • Detect using HRP-conjugated secondary detection system

  • Analyze using flow cytometry or dedicated bead array readers

These approaches enable comprehensive pathway analysis rather than isolated protein measurements, providing mechanistic insights into glutathione homeostasis regulation .

How are emerging technologies enhancing the utility of GSS Antibody, HRP conjugated in research?

Emerging technologies are expanding applications:

• Single-cell protein analysis:

  • Integration with microfluidic platforms

  • Adaptation for mass cytometry (CyTOF)

  • Development of highly sensitive, low-volume assays

• Advanced imaging modalities:

  • Super-resolution microscopy compatibility

  • Whole-tissue clearing and 3D imaging

  • Intravital microscopy for in vivo GSS dynamics

• Automation and high-throughput screening:

  • Robotics-compatible assay formats

  • Machine learning-based image analysis

  • Integration with automated liquid handling systems

• Enhanced conjugation technologies:

  • Site-specific conjugation for improved HRP orientation

  • Alternative enzyme conjugates with improved stability

  • Quantum dot conjugation for enhanced sensitivity and multiplexing

The continued refinement of recombinant antibody technologies and conjugation methods promises to further enhance the utility of GSS detection tools in research applications .

What are the current limitations of GSS Antibody, HRP conjugated and potential solutions?

Current limitations and potential solutions include:

• Temperature sensitivity:

  • Limitation: HRP activity is temperature-sensitive

  • Solution: Development of thermostable HRP variants or alternative enzymes

• Batch-to-batch variability:

  • Limitation: Polyclonal antibodies show lot variation

  • Solution: Recombinant antibody production with consistent properties

• Limited shelf-life:

  • Limitation: HRP activity decreases over time

  • Solution: Improved stabilization buffers and lyophilization technologies

• Detection range constraints:

  • Limitation: Dynamic range of 40 pM to 5 nM

  • Solution: Enhanced signal amplification systems for broader quantitation range

• Cross-reactivity issues:

  • Limitation: Potential binding to closely related proteins

  • Solution: Epitope-specific monoclonal antibody development