GST Mouse Antibody

What is GST and why are GST Mouse Antibodies important in protein research?

GST (Glutathione S-transferase) is a 26kDa protein present in both eukaryotes and prokaryotes, where it catalyzes various reactions. In research settings, GST is widely used in gene fusion systems to create tagged proteins that can be readily purified through their high affinity for glutathione . GST Mouse Antibodies are monoclonal antibodies raised in mice that specifically recognize the GST protein or GST-tagged fusion proteins.

These antibodies are particularly valuable because they:

• Allow for detection of fusion proteins during purification processes

• Help monitor cleavage of GST from proteins of interest

• Enable tracking of GST-tagged proteins in various experimental contexts

• Support protein-protein interaction studies using GST-fusion systems

The GST-fusion protein system has become a fundamental tool in molecular biology as proteins can be purified to homogeneity in a single step, as the GST portion binds tightly to immobilized glutathione .

How do I choose the appropriate GST Mouse Antibody for my specific research application?

Selecting the optimal GST Mouse Antibody depends on several key considerations:

Specificity requirements:

• Determine whether you need an antibody that recognizes Schistosoma japonicum GST specifically or one with broader reactivity

• Some antibodies, like the #21 clone, specifically react with S. japonicum GST protein and GST-fused proteins

• Others, such as GST.B6 clone, can recognize both native and denatured forms of purified GST or GST fusion proteins

Experimental application:
For each application, consider these recommended dilution ranges:

Antibody format:

• Consider whether you need a conjugated or unconjugated antibody

• Most GST Mouse Antibodies are available in unconjugated form and require appropriate secondary antibodies

What are the optimal storage conditions for maintaining GST Mouse Antibody activity?

To maintain optimal activity of GST Mouse Antibodies, follow these evidence-based storage guidelines:

• Store shipped antibodies at 4°C upon delivery

• For long-term storage, aliquot the antibody and store at -20°C

• Avoid repeated freeze/thaw cycles as they can compromise antibody function

• Most GST Mouse Antibodies are supplied in stabilizing buffers containing preservatives:

  • Typically in PBS with 50% glycerol

  • Some include 0.02% sodium azide and 1.5% BSA at pH 7.3

  • Others may be in 10mM PBS at pH 7.2 with 0.05% NaN₃

When working with the antibody, thaw aliquots completely before use and keep on ice during experimental procedures. Return unused antibody to appropriate storage conditions immediately after use.

What is the recommended protocol for using GST Mouse Antibody in Western blot applications?

Based on validated protocols from multiple sources, here is a comprehensive Western blot methodology using GST Mouse Antibody:

Sample preparation:

• Load 2-5 μg of purified GST fusion protein or cell lysate containing GST-tagged proteins per lane

• Use appropriate positive controls (purified GST protein) and negative controls

Electrophoresis and transfer:

• Separate proteins using standard SDS-PAGE (10-12% gels are typically suitable for GST fusion proteins)

• Transfer to nitrocellulose or PVDF membrane using standard protocols

Blocking and antibody incubation:

• Block membrane with 3-5% nonfat dry milk in TBST for 1 hour at room temperature

• Dilute primary GST Mouse Antibody according to manufacturer's recommendations:

  • Typically 1:1000-1:3000 for Western blot applications

  • Incubate overnight at 4°C or 1-2 hours at room temperature with gentle agitation

• Wash membrane 3-5 times with TBST (5 minutes each)

• Incubate with appropriate HRP-conjugated secondary antibody:

  • Anti-Mouse IgG secondary antibodies at 1:10,000-1:20,000 dilution

  • Examples include Goat Anti-Mouse IgG (H&L) [HRP] or equivalent

• Wash membrane 3-5 times with TBST (5 minutes each)

Detection:

• Develop using ECL detection reagents

• Exposure times typically range from 1-30 seconds depending on protein abundance

The figure from ABclonal shows successful Western blot detection of overexpressed GST protein using their Mouse anti GST-Tag mAb (AE001) with clear bands at approximately 27 kDa .

How can I optimize immunoprecipitation protocols using GST Mouse Antibody?

For successful immunoprecipitation of GST-tagged proteins, follow this optimized protocol:

Pre-clearing (optional but recommended):

• Mix cell lysate with protein A/G beads for 1 hour at 4°C

• Centrifuge and collect supernatant

Antibody-antigen binding:

• Dilute GST Mouse Antibody in IP buffer at 1:50-1:200

• For most effective results, use 2-5 μg antibody per 500 μg of total protein

• Incubate with pre-cleared lysate overnight at 4°C with gentle rotation

Immunoprecipitation:

• Add 30-50 μl of protein A/G beads to the lysate-antibody mixture

• Incubate for 2-4 hours at 4°C with gentle rotation

• Centrifuge at 2500-3000 rpm for 2-3 minutes at 4°C

• Carefully remove supernatant

Washing and elution:

• Wash beads 3-5 times with cold IP buffer

• Elute bound proteins by adding SDS-PAGE sample buffer and heating at 95°C for 5 minutes

• Analyze by Western blotting using standard protocols

When troubleshooting IP experiments, consider:

• Increasing antibody concentration for low-abundance targets

• Adjusting salt concentration in wash buffers to reduce non-specific binding

• Using crosslinking methods if the interaction is transient or weak

What controls should be included when working with GST Mouse Antibody?

Proper experimental controls are critical for valid interpretation of results with GST Mouse Antibody:

Positive controls:

• Purified GST protein or GST-tagged fusion protein with known expression

• Lysates from cells transfected with GST-expressing vectors

• Commercial GST protein standards

Negative controls:

• Non-transfected cell lysates (for recombinant expression systems)

• Isotype control: Mouse IgG of the same isotype (e.g., IgG1 or IgG2a depending on the antibody)

• For IP experiments: beads-only control and irrelevant antibody control

Validation controls:

• When evaluating antibody specificity, test against N-terminal, C-terminal, and internal GST-tagged fusion proteins as shown in Figure 1 of GenScript data

• For quantitative assays, include calibration standards with known amounts of GST protein

Inclusion of appropriate controls allows for accurate interpretation of results and troubleshooting of potential issues with specificity or sensitivity.

How can GST Mouse Antibody be used to investigate different GST isoforms in subcellular compartments?

GST Mouse Antibody can be used in conjunction with subcellular fractionation techniques to identify and characterize GST isoforms in different cellular compartments. This approach has been validated in research examining mitochondrial GST isoforms:

Methodology:

• Perform subcellular fractionation to isolate mitochondria and other cellular compartments

• Enrich GST proteins using GSH affinity chromatography from isolated fractions

• Separate proteins using 2D electrophoresis or SDS-PAGE

• Identify GST isoforms using mass spectrometry (MALDI TOF/TOF MS or ESI MS/MS)

• Confirm findings using Western blotting with GST Mouse Antibody

This comprehensive approach has successfully identified five GST isoforms (alpha3, mu1, pi1, kappa1, and zeta1) in mouse liver mitochondria, with GST kappa1 being specifically localized to mitochondria .

Distribution of GST isoforms across tissues:
Research has demonstrated that GST isoforms are distributed differently across tissues:

Note: Relative abundance indicated by number of check marks (✓)

This approach can be applied to study GST distribution in various disease models, as demonstrated by the comparison between normal and diabetic mice .

How can I quantitatively assess GST-tagged protein expression using GST Mouse Antibody?

Quantitative assessment of GST-tagged proteins requires careful calibration and standardization:

Calibration method:

• Prepare a dilution series of purified recombinant GST protein at known concentrations

• Perform Western blotting with GST Mouse Antibody using identical conditions for standards and samples

• Generate calibration curves correlating protein concentrations with immunoblot signal intensities

• Quantify unknown samples against this standard curve

This approach has been successfully applied in research studying GST isoforms in normal and diabetic mice .

Statistical considerations:

• Run at least three biological replicates for statistical validity

• Normalize signal intensities to appropriate loading controls

• Use image analysis software (e.g., ImageQuant TL) to quantify band intensities

• Apply appropriate statistical tests (e.g., Student's t-test) with significance threshold of p < 0.05

Example calculation:
For comparing GST expression between experimental conditions:

• Calculate the relative intensity of GST signals in each sample

• Normalize to control samples

• Derive mean ± SD from replicate experiments

• Perform statistical analysis to determine significant differences

This quantitative approach provides robust data on changes in GST expression under different experimental conditions.

What strategies can improve detection of differentially positioned GST tags in fusion proteins?

GST tags can be positioned at the N-terminus, C-terminus, or internally within fusion proteins, which may affect antibody recognition. Based on experimental evidence, consider these strategies:

Optimization approaches:

• Test antibody recognition against different GST tag positions as demonstrated in Figure 1 from GenScript data, which shows successful detection of N-terminal, internal, and C-terminal GST-tagged fusion proteins

• Adjust antibody concentration based on tag position and accessibility

• Consider protein conformation effects on epitope accessibility

Recommended antibodies:
Some GST Mouse Antibodies have been specifically validated for detection of differentially positioned tags:

• THETM GST Antibody (GenScript, A00865) has demonstrated affinity for all three positions (N-terminal, internal, C-terminal)

• When selecting antibodies, prioritize those with validation data showing detection at multiple positions

Application-specific considerations:
For detecting GST-tagged proteins in complex samples:

• Western blotting: Denaturing conditions may reduce position-dependent recognition differences

• Immunoprecipitation: Tag position may significantly affect antibody accessibility in native conditions

• Immunofluorescence: Consider whether tag position affects subcellular localization signals

What are common challenges when using GST Mouse Antibody and how can they be resolved?

Problem: Weak or no signal in Western blot
Potential causes and solutions:

• Insufficient antibody concentration: Increase primary antibody concentration (try 1:1000 instead of 1:3000)

• Inadequate protein loading: Increase sample concentration to 2-5 μg per lane

• Poor transfer: Optimize transfer conditions or validate with Ponceau S staining

• Protein degradation: Add protease inhibitors during sample preparation

• Secondary antibody mismatch: Ensure secondary antibody recognizes mouse IgG (host species)

Problem: High background in Western blot
Potential causes and solutions:

• Insufficient blocking: Increase blocking time or concentration (try 5% instead of 3% milk)

• Excessive antibody concentration: Reduce primary antibody concentration

• Inadequate washing: Increase number or duration of wash steps

• Non-specific binding: Add 0.1-0.5% Tween-20 to antibody dilution buffer

Problem: Multiple bands in Western blot
Potential causes and solutions:

• Protein degradation: Add protease inhibitors and keep samples cold

• Antibody cross-reactivity: Validate antibody specificity with positive and negative controls

• Post-translational modifications: Consider phosphatase treatment if modification is suspected

• GST isoforms: Confirm if multiple bands represent different GST isoforms as multiple isoforms have been identified in tissues

Problem: Poor immunoprecipitation efficiency
Potential causes and solutions:

• Insufficient antibody: Increase antibody amount (use closer to 1:50 dilution)

• Weak antibody-protein interaction: Optimize binding conditions (buffer composition, incubation time)

• Poor bead binding: Pre-clear lysate or increase bead amount

• Detergent interference: Adjust detergent concentration in lysis and wash buffers

How should experimental conditions be modified when working with low-abundance GST-tagged proteins?

When working with low-abundance GST-tagged proteins, consider these evidence-based modifications:

Sample preparation enhancements:

• Increase starting material (2-3x standard amount)

• Use GSH-based enrichment prior to analysis

• Consider using proteasome inhibitors during cell lysis to prevent protein degradation

Detection optimization:

• Use more sensitive detection methods (e.g., chemiluminescent substrates with femtogram sensitivity)

• Increase exposure time during imaging (but monitor background)

• Reduce antibody dilution (use 1:1000 instead of 1:3000)

• Consider signal amplification systems like biotinylated secondary antibodies with streptavidin-HRP

Immunoprecipitation modifications:

• Increase antibody:lysate ratio (use 1:50 instead of 1:200 dilution)

• Extend antibody-antigen incubation time to overnight at 4°C

• Reduce wash stringency to preserve weak interactions

• Elute with reduced sample volume to concentrate protein

Western blot adaptations:

• Load maximum protein amount per lane (up to 50-100 μg total protein)

• Use higher percentage acrylamide gels (12-15%) to better resolve and concentrate small proteins

• Extend transfer time for complete protein transfer

• Use PVDF membranes (higher protein binding capacity than nitrocellulose)

How can GST Mouse Antibody be used in proteomic studies of GST isoforms?

GST Mouse Antibody can be leveraged in sophisticated proteomic workflows to characterize GST isoforms across tissues and disease states:

Multi-stage proteomic approach:

• Affinity enrichment using GSH chromatography to isolate GST proteins

• Protein separation using 2DE or SDS-PAGE

• Mass spectrometry identification (MALDI TOF/TOF MS or ESI MS/MS)

• Verification by Western blotting with GST Mouse Antibodies

This comprehensive workflow has been successfully used to identify five GST isoforms in mouse liver mitochondria .

Quantitative proteomic applications:

• Create calibration curves using recombinant GST proteins of known concentration

• Compare GST isoform expression between normal and disease states (e.g., diabetes)

• Normalize signal intensity against appropriate controls

• Perform statistical analysis to identify significant differences

Comparative tissue analysis:
GST Mouse Antibody can be used to evaluate tissue-specific distribution of GST isoforms:

• Different tissues (e.g., liver, kidney, heart) show variable GST isoform profiles

• GST kappa1 shows consistent presence across tissues as a mitochondria-specific isoform

• GST pi1 demonstrates notably higher abundance in liver mitochondria compared to other tissues

This approach provides deeper insights into the biological significance of GST distribution and expression changes in various physiological and pathological conditions.

What considerations are important when designing GST pulldown assays using GST Mouse Antibody?

GST pulldown assays are valuable for studying protein-protein interactions. When designing these experiments with GST Mouse Antibody, consider these critical factors:

Assay design optimization:

• Express GST-tagged bait protein in appropriate expression system

• Include proper controls:

  • GST-only control to identify non-specific binding partners

  • Empty vector control

  • Irrelevant GST-fusion protein control

• Pre-clear lysates to reduce background

• Optimize salt and detergent concentrations to balance specificity and sensitivity

Antibody selection and validation:

• Confirm antibody specificity for your particular GST fusion construct

• Validate using Western blotting before pulldown experiments

• Verify recognition of both free GST and GST-fusion proteins

Technical recommendations:

• For binding reactions:

  • Use freshly prepared GST-fusion proteins where possible

  • Maintain physiologically relevant buffer conditions

  • Include protease inhibitors and phosphatase inhibitors if studying phosphorylation-dependent interactions

• For washing:

  • Optimize wash stringency to preserve specific interactions while removing non-specific binding

  • Consider using decreasing salt gradients in sequential washes

Detection strategies:

• Western blotting with antibodies against interacting partners

• Mass spectrometry for unbiased identification of novel interacting proteins

• Complementary techniques (e.g., reciprocal co-IP) to confirm interactions

How does the choice of GST Mouse Antibody clone affect experimental outcomes?

Different GST Mouse Antibody clones have distinct characteristics that can significantly impact experimental results:

Clone-specific properties comparison:

Application-specific considerations:

• Western blotting: Antibodies recognizing denatured epitopes perform better

• Immunoprecipitation: Antibodies with high affinity for native conformation are preferred

• Immunofluorescence: Accessibility of epitope in fixed/permeabilized cells is crucial

Validation approaches:

• Compare multiple clones side-by-side with standardized conditions

• Test recognition of different GST fusion constructs (N-terminal, internal, C-terminal)

• Evaluate performance across different applications before committing to large-scale experiments

Selecting the optimal clone for specific experimental needs can significantly improve sensitivity, specificity, and reproducibility of results.