GST Monoclonal antibody

What is a GST monoclonal antibody and how does it function in research applications?

GST monoclonal antibodies are specifically designed to recognize and bind to Glutathione S-transferase, a family of cytosolic dimeric isoenzymes that play an important role in detoxification by catalyzing the conjugation of reduced glutathione to various compounds . In research settings, these antibodies primarily detect GST-tagged fusion proteins, which are common in recombinant protein expression systems .

GST is highly conserved across most organisms, making it an ideal tag for protein expression systems . These antibodies function by specifically binding to the GST portion of fusion proteins, allowing researchers to detect, purify, or study the interaction of the tagged protein of interest . Different monoclonal antibodies target specific epitopes on the GST protein, with some antibodies specifically recognizing GST from Schistosoma japonicum (as with antibody clone 6G9) .

The antibodies are produced through various methods, including traditional hybridoma technology (mouse, rat antibodies) and newer recombinant technologies (human, rabbit antibodies), each offering specific advantages for different research applications .

What are the primary applications of GST monoclonal antibodies in laboratory research?

GST monoclonal antibodies have several important applications in modern laboratory research:

For protein-protein interaction studies, particularly using HTRF (Homogeneous Time-Resolved Fluorescence) technology, GST antibodies conjugated with fluorescent labels (XL665 or Eu cryptate) are especially valuable . These labeled antibodies enable researchers to study a wide variety of interactions, including protein/protein, protein/peptide, protein/DNA, and protein/small molecule interactions, across a broad range of affinity constants from picomolar to low millimolar .

How do different host species affect GST antibody performance in experimental settings?

The host species in which a GST monoclonal antibody is produced significantly influences its performance characteristics and experimental applications:

Recombinant chimeric antibodies, such as those offered by BiCell Scientific, combine the Fv domain of a mouse hybridoma clone with the Fc domain of a selected species (mouse, rat, or rabbit) . This approach allows researchers to retain original binding characteristics while gaining greater species flexibility, which can be particularly important when designing complex multi-antibody experiments or working with tissues that may cross-react with certain antibody species .

What are the optimal dilution ratios for GST monoclonal antibodies in different applications?

Optimal dilution ratios for GST monoclonal antibodies vary by application and specific antibody clone. Based on manufacturer recommendations:

It's important to note that optimal dilutions should be determined empirically for each experimental system . Factors that may influence the optimal dilution include sample type (cell lysates versus purified proteins), detection system (chemiluminescent versus fluorescent), and antibody characteristics (affinity and format) .

Always perform a dilution series during initial optimization to determine the concentration that provides the best signal-to-noise ratio for your specific experimental conditions.

How should researchers validate the specificity of GST antibodies for their specific fusion proteins?

Validating the specificity of GST antibodies for specific fusion proteins is a critical step in ensuring reliable experimental results. A comprehensive validation approach should include:

• Positive and negative controls:

  • Include purified GST protein and known GST-fusion proteins (e.g., Alpha-actinin-4-GST, AKT1/PKBa-GST, or CDK7-GST) as positive controls

  • Include non-GST tagged versions of your protein of interest and lysates from non-transfected cells as negative controls

  • Express and purify GST alone under the same conditions as a tag-only control

• Multiple antibody evaluation:

  • Test different GST antibody clones (e.g., AbD03937, 6G9, EPR4236)

  • Compare antibodies from different host species or production methods

  • Document any differences in recognition patterns

• Sequential analysis techniques:

  • Perform Western blot analysis to confirm a single band of expected molecular weight

  • Consider immunoprecipitation followed by mass spectrometry to verify pulled-down protein identity

  • Use immunofluorescence co-localization to compare with alternative detection methods

• Epitope characterization:

  • Determine if the antibody recognizes specific GST variants (e.g., from Schistosoma japonicum versus other sources)

  • Test for cross-reactivity with endogenous GST family members if relevant to your experimental system

  • For recombinant systems, confirm no cross-reactivity with host cell proteins

For advanced validation, particularly with novel fusion proteins, consider using orthogonal methods such as mass spectrometry or alternative tagging systems to confirm the identity and behavior of your GST-fusion protein.

What controls are essential when using GST monoclonal antibodies in various research applications?

Essential controls when using GST monoclonal antibodies include:

• Positive controls:

  • Purified GST protein to confirm antibody functionality

  • Well-characterized GST-fusion proteins (such as Alpha-actinin-4-GST, AKT1/PKBa-GST, or CDK7-GST)

  • Previously validated samples known to contain GST-tagged protein

• Negative controls:

  • Non-transfected cells or tissues (for recombinant expression experiments)

  • Samples expressing the untagged version of the protein of interest

  • Samples expressing an irrelevant GST-fusion protein

• Antibody controls:

  • Isotype control antibody (same isotype as the GST antibody but with irrelevant specificity)

  • Secondary antibody only (to assess background from the detection system)

  • Pre-adsorption control (GST antibody pre-incubated with purified GST protein)

• Method-specific controls:

  For Western blot:

  • Molecular weight markers to confirm expected size

  • Loading controls (housekeeping proteins)

  • Non-reducing vs. reducing conditions if relevant

  For immunofluorescence:

  • DAPI nuclear counterstain

  • Cytoskeletal markers (e.g., anti-tubulin at 1/1000)

  • Untransfected cells processed identically

  For protein-protein interaction studies:

  • Known interacting partners as positive controls

  • Non-interacting proteins as negative controls

  • Competition with untagged proteins to confirm specificity

A systematic approach to controls not only validates antibody specificity but also helps troubleshoot experimental issues. Documentation of all controls is essential for publication-quality research and reproducibility.

How can GST monoclonal antibodies be optimized for protein-protein interaction studies?

Optimizing GST monoclonal antibodies for protein-protein interaction studies involves several strategic considerations:

• Selection of appropriate antibody format and label:

  • For HTRF (Homogeneous Time-Resolved Fluorescence) assays, specific labeled antibodies such as MAb Anti GST-XL665 (acceptor) or MAb Anti GST-Eu cryptate (donor) are essential

  • The choice between donor and acceptor labels depends on the experimental design and the other interaction partners

• Assay design optimization:

  • In a typical HTRF interaction assay protocol using a 20 μL final volume:
    a. Dispense the 2 interaction partners (10 μL)
    b. Incubate to allow interaction
    c. Add labeled antibodies (5 μL each of donor and acceptor)
    d. Incubate and read

• Partner tagging strategy:

  • When one partner is GST-tagged, the second partner can be:

    • Non-tagged (detected with specific antibody)

    • Biotinylated (detected with streptavidin-conjugates)

    • Tagged with another tag (detected with corresponding anti-tag antibody)

    • Fc-fused (detected with anti-species or protein A reagents)

• Controls and validation:

  • Include appropriate non-interacting GST-tagged proteins as negative controls

  • Use known interacting partners as positive controls

  • Consider competition assays with untagged proteins to confirm specificity

For maximum sensitivity in detecting interactions with wide-ranging affinities (from picomolar to low millimolar), HTRF technology combined with appropriate GST antibody conjugates has proven particularly effective .

How do conjugated GST antibodies (XL665, Eu cryptate) differ in sensitivity and performance?

Conjugated GST antibodies show distinct performance characteristics based on their specific labels:

Sensitivity comparison:

• Detection limits:

  • HTRF with XL665/Eu cryptate pairs can detect interactions with affinity constants ranging from picomolar to low millimolar

  • Time-resolved detection significantly improves signal-to-noise ratios compared to conventional fluorescence

• Signal stability:

  • XL665 and Eu cryptate conjugates show excellent signal stability over time

  • Long fluorescence lifetimes allow for time-resolved measurements, eliminating short-lived background fluorescence

• Application-specific performance:

  • For protein-protein interactions in HTRF format:

    • The labeled antibody directly binds to the GST-tagged partner

    • Signal intensity is proportional to the binding of the two partners

    • The assay protocol typically uses a 20 μL final volume

How do recombinant GST monoclonal antibodies compare to traditional hybridoma-derived antibodies?

Recombinant GST monoclonal antibodies offer several distinctive advantages compared to traditional hybridoma-derived antibodies:

Key advantages of recombinant antibodies include:

• Defined sequence and structure:

  • The amino acid sequence is known and can be verified

  • No contamination with irrelevant antibodies

  • Possibility to optimize codon usage for expression system

• Engineering possibilities:

  • Humanization for therapeutic applications

  • Modification of binding characteristics

  • Addition of specific tags or conjugation sites

  • Creation of bispecific or multispecific formats

• Quality and reproducibility:

  • Consistent quality from lot to lot

  • Defined affinity and specificity

  • Long-term supply security

Specific examples from the search results:

• Recombinant rabbit monoclonal EPR4236 is produced using RabMAb® technology, offering high batch-to-batch consistency and animal-free production

• BiCell Scientific's recombinant GST antibody is a chimeric antibody consisting of the Fv domain sequence of mouse hybridoma clone N100/13 and the Fc domain sequence of mouse, rat or rabbit, providing flexibility in experimental design

How can researchers address non-specific binding issues with GST monoclonal antibodies?

Non-specific binding is a common challenge when working with GST monoclonal antibodies. Here are comprehensive strategies to address this issue:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Increase blocking time or concentration

  • Consider adding 0.1-0.5% Tween-20 to blocking and washing buffers

• Antibody optimization:

  • Titrate antibody concentration (higher dilutions often reduce non-specific binding)

  • Try different GST antibody clones (e.g., switch from mouse to rabbit or rat antibodies)

  • Consider using F(ab) or F(ab')2 fragments instead of whole IgG

  • For recombinant systems, consider HuCAL Fab bivalent format (like AbD03937)

• Sample preparation improvements:

  • Pre-clear lysates with Protein G/A beads before immunoprecipitation

  • Add more stringent washing steps with increased salt concentration

  • Use freshly prepared samples to minimize protein degradation

  • Include protease inhibitors in lysis buffers

• Buffer optimization:

  • Add carrier proteins (e.g., 0.1% BSA) to dilution buffers

  • Optimize salt concentration in washing buffers

  • Consider adding mild detergents (0.05-0.1% NP-40 or Triton X-100)

• Experiment-specific approaches:

  For Western blot:

  • Reduce exposure time

  • Optimize transfer conditions (time, buffer, voltage)

  • Consider membrane type (PVDF versus nitrocellulose)

  For immunofluorescence:

  • Optimize fixation (4% PFA) and permeabilization conditions (0.1% Triton X-100)

  • Increase washing steps and duration

  • Use confocal microscopy for better signal discrimination

  For HTRF assays:

  • Optimize the donor/acceptor ratio

  • Include appropriate negative control interactions

  • Test for spectral overlap and adjust calculations accordingly

Systematic documentation of optimization steps will help establish reliable protocols for specific experimental systems.

How should conflicting results between different GST antibody clones be interpreted?

When different GST antibody clones produce conflicting results, systematic interpretation and troubleshooting are essential:

• Epitope differences analysis:

  • Different clones (e.g., AbD03937, 6G9, EPR4236, S-tag-05) recognize different epitopes on GST

  • Map the specific epitopes recognized by each antibody if information is available

  • Consider whether the fusion protein might mask certain epitopes

  • Evaluate if epitopes are conserved across GST variants (Schistosoma japonicum vs. other sources)

• Antibody format considerations:

  • Compare the formats of conflicting antibodies:

    • Whole IgG vs. Fab fragments

    • Different species origins (rabbit, mouse, rat, human)

    • Monoclonal vs. recombinant

    • Different conjugates (unconjugated vs. XL665 or Eu cryptate-labeled)

• Experimental condition analysis:

  • Determine if differences appear under specific conditions:

    • Native vs. denaturing conditions

    • Reducing vs. non-reducing environments

    • Different buffer compositions

    • Various sample types (purified protein vs. cell lysate)

• Validation with orthogonal methods:

  • Confirm protein identity and presence using non-antibody methods:

    • Mass spectrometry

    • Activity assays for GST

    • Alternative tag detection if available

    • mRNA expression confirmation

• Resolution strategies:

  Conflict Type	Resolution Approach
  Presence/absence conflict	- Use enrichment strategies (IP before detection) - Increase sample concentration - Try alternative sample preparation methods
  Signal intensity differences	- Perform titration experiments with both antibodies - Compare to known standards - Normalize to total protein
  Molecular weight differences	- Check for post-translational modifications - Verify complete denaturation - Confirm protein identity by mass spectrometry
  Subcellular localization differences	- Use fractionation to verify locations - Perform co-localization with known markers - Test fixation artifacts with different methods

When interpreting conflicting results, remember that some discrepancies may reveal important biological insights rather than representing technical failures. The key is to systematically investigate the source of differences and determine which results most accurately reflect the biological reality.

What are the considerations for using GST antibodies in HTRF (Homogeneous Time-Resolved Fluorescence) assays?

HTRF assays with GST antibodies require specific considerations for optimal performance:

• Assay design fundamentals:

  • Basic principle: In HTRF interaction assays, one partner is labeled with a donor fluorophore and the other with an acceptor

  • Signal generation: FRET (Fluorescence Resonance Energy Transfer) occurs when the two partners interact, generating a specific signal

  • Measurement: The ratio of acceptor to donor emission indicates interaction strength

• Partner labeling strategy:

  • For GST-tagged proteins:

    • Use MAb Anti GST-XL665 (acceptor) when the other partner is labeled with donor

    • Use MAb Anti GST-Eu cryptate (donor) when the other partner is labeled with acceptor

  • For the interaction partner:

    • Direct labeling with compatible HTRF fluorophore

    • Use of biotinylated partner with streptavidin-conjugated fluorophore

    • Use of tag-specific antibody labeled with compatible fluorophore

    • Use of anti-species or Protein A reagents for Fc-fused partners

• Protocol optimization:

  • Typical HTRF protocol for 20 μL final volume:

    1. Dispense the two interaction partners (10 μL)

    2. Incubate to allow interaction

    3. Add labeled antibodies (5 μL each of donor and acceptor antibodies)

    4. Incubate and read in a compatible fluorescence reader

• Reagent considerations:

  • Antibody quantity: Based on assay points (e.g., 1,000 or 5,000 assay points per package)

  • Buffer compatibility: Ensure buffer components don't interfere with fluorescence

  • Protein concentration: Optimize to be within the linear range of detection

  • Incubation times: Typically longer for weaker interactions

• Technical aspects of HTRF detection:

  • Time-resolved measurement: Typically 50-150 μs delay after excitation

  • Dual wavelength detection: Measure both donor (620 nm) and acceptor (665 nm) emissions

  • Ratiometric calculation: Divide acceptor signal by donor signal to normalize for assay variations

HTRF technology with GST antibodies is particularly valuable because it can detect interactions across a broad range of affinities (from picomolar to low millimolar) and can be applied to various interaction types, including protein/protein, protein/peptide, protein/DNA, protein/RNA, protein/carbohydrate, and protein/small molecule interactions .

What are the advantages of using GST monoclonal antibodies in cellular versus biochemical assay formats?

GST monoclonal antibodies show distinctive performance characteristics in cellular versus biochemical assay formats:

In cellular formats, GST antibodies like the rabbit monoclonal EPR4236 have been successfully used in immunofluorescence studies of transfected cells . When performing ICC/IF with GST antibodies, key considerations include:

• Cell preparation:

  • Fixation method (commonly 4% PFA)

  • Permeabilization (often 0.1% Triton X-100)

• Antibody parameters:

  • Working dilution (typically 1/100 for ICC/IF versus 1/1000 for WB)

  • Incubation time and temperature

• Detection strategy:

  • Secondary antibody selection (e.g., Alexa Fluor 488 goat anti-rabbit)

  • Counterstaining (e.g., DAPI for nuclei, anti-tubulin for cytoskeleton)

• Controls:

  • Non-transfected cells or cells expressing non-GST tagged proteins

  • Secondary antibody only controls

For biochemical formats like HTRF assays, GST antibodies conjugated to donor or acceptor molecules provide excellent sensitivity for detecting protein-protein interactions across a broad range of affinities, from picomolar to low millimolar .