GSC Antibody, Biotin conjugated

What is GST Antibody, Biotin Conjugated and how is it produced?

GST Antibody, Biotin Conjugated is a polyclonal antibody generated in goats that recognizes Glutathione-S-Transferase (GST) from Schistosoma japonicum. This research reagent is prepared through a precise multi-step process involving immunoaffinity chromatography using GST coupled to agarose beads, followed by solid phase adsorption to remove unwanted reactivities and extensive dialysis against a stabilizing buffer solution. The purified antibody is then conjugated with biotin molecules to enable detection via the high-affinity biotin-(strept)avidin interaction system .

Immunoelectrophoresis testing confirms its specificity, showing a single precipitin arc against anti-biotin, anti-Goat Serum, and both purified and partially purified GST from Schistosoma japonicum . This production methodology ensures high specificity while maintaining robust biotin availability for downstream detection applications.

What is the scientific basis of the biotin-(strept)avidin system in immunoassays?

The biotin-(strept)avidin system represents one of the strongest non-covalent interactions known in biological systems, with dissociation constants (KD) ranging from 10^-14 to 10^-15, which is approximately 10^3 to 10^6 times stronger than typical antibody-antigen interactions . This extraordinary binding affinity provides several advantages in immunoassay design:

• Signal amplification capability, particularly valuable for detecting low-abundance analytes

• Reduction in required procedural steps, enabling more rapid quantitation

• Exceptional stability against proteolytic enzymes, temperature and pH extremes, and harsh chemical conditions

• Versatility in assay architecture design and implementation

The system's remarkable stability and specificity make it particularly suitable for isolating and amplifying signals in complex biological matrices like serum or plasma, where capturing an analyte of interest with high specificity is critical .

What are the validated applications for GST Antibody, Biotin Conjugated?

GST Antibody, Biotin Conjugated has been validated for several research applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): Primary application with demonstrated effectiveness for detecting and quantifying GST-tagged proteins

• IF (Immunofluorescence): Validated for cellular localization studies of GST-tagged proteins

• Immunoblotting: Suitable for western blot applications when used with appropriate detection systems

• Immunohistochemistry: Effective for tissue section analysis when GST-tagged proteins need to be visualized

• Immunomicroscopy: Applicable for microscopic visualization of GST-tagged proteins in various sample types

Additionally, this antibody can be used in any antibody-based assay that employs streptavidin or avidin conjugates and requires consistent lot-to-lot performance. The optimal concentration for each application should be empirically determined by the researcher based on specific experimental conditions .

How does the binding affinity of biotin-(strept)avidin compare quantitatively to other immobilization systems used in immunoassays?

The biotin-(strept)avidin system exhibits extraordinary binding affinity compared to other commonly used immobilization systems in immunoassays. The table below presents a quantitative comparison:

This exceptional affinity makes the biotin-(strept)avidin system particularly valuable for applications requiring high sensitivity, strong immobilization, and resistance to washing steps . The 10^3-10^6-fold stronger binding compared to typical antibody-antigen interactions contributes to the system's ability to maintain stable complexes under stringent experimental conditions that would disrupt other binding interactions.

What mechanisms underlie biotin interference in immunoassays employing GST Antibody, Biotin Conjugated?

Biotin interference represents a significant concern in immunoassays utilizing the biotin-(strept)avidin system, including those employing GST Antibody, Biotin Conjugated. The interference occurs through several mechanisms:

• Competitive inhibition: High levels of free biotin (from dietary supplements or endogenous sources) compete with biotinylated reagents for binding to (strept)avidin sites, reducing the binding of biotinylated GST antibodies to their intended targets .

• Assay architecture effects: The impact of biotin interference varies based on assay design:

  • In sandwich assays using biotinylated capture antibodies, interference typically leads to falsely decreased results

  • In competitive assays, interference often produces falsely elevated results

• Concentration-dependent effects: The severity of interference correlates with free biotin concentration in the sample, with high-dose biotin supplements (5-10 mg/day) posing greater risk than typical multivitamin levels .

The FDA has issued safety notices regarding this issue, highlighting the potential for biotin interference to generate misleading diagnostic results, which could compromise research data validity when using GST Antibody, Biotin Conjugated in similar experimental systems .

What factors determine cross-reactivity profiles of GST Antibody, Biotin Conjugated?

The cross-reactivity profile of GST Antibody, Biotin Conjugated is influenced by multiple experimental and intrinsic factors:

• Source organism specificity: The antibody is raised against GST from Schistosoma japonicum, making it optimally reactive with this specific protein. Cross-reactivity with GST from other sources may occur but varies in intensity .

• Epitope conservation: The degree of amino acid sequence homology between GST variants affects cross-recognition. Highly conserved regions will promote cross-reactivity, while divergent regions reduce it.

• Production methodology: The immunoaffinity chromatography and solid phase adsorption techniques used during production help minimize unwanted reactivities by removing antibodies that bind to non-target epitopes .

• Conjugation effects: The biotin conjugation process can potentially affect antigen-binding properties if biotin molecules are attached near the antigen-binding site.

• Sample preparation conditions: Buffer composition, pH, and the presence of detergents or reducing agents can alter epitope accessibility and antibody binding characteristics.

Researchers should validate cross-reactivity experimentally when working with GST variants from organisms other than Schistosoma japonicum, particularly when precise target specificity is critical for experimental outcomes .

What are the optimal storage and handling protocols for GST Antibody, Biotin Conjugated?

To maintain optimal reactivity and stability of GST Antibody, Biotin Conjugated, researchers should adhere to the following storage and handling guidelines:

• Short-term storage: Store unopened vial at 4°C prior to reconstitution .

• Reconstitution procedure:

  • Restore lyophilized antibody with deionized water or equivalent

  • Allow complete dissolution before use

  • Avoid introducing contaminants during reconstitution

• Long-term storage:

  • For extended storage, aliquot reconstituted antibody into working volumes

  • Freeze aliquots at -20°C or below

  • Avoid repeated freeze-thaw cycles that can denature antibody proteins

• Working solution preparation:

  • Centrifuge product if not completely clear after standing at room temperature

  • Dilute only immediately before use to maintain stability

  • Prepare working solutions in appropriate buffer systems

• Stability considerations:

  • Product remains stable for several weeks at 4°C as an undiluted liquid

  • Expected shelf life is one year from receipt date when stored properly

  • Monitor for signs of degradation such as precipitation or loss of activity

Proper adherence to these guidelines ensures maximum antibody performance and reproducibility across experiments.

How do I design an immunoassay using the biotin-(strept)avidin system with GST Antibody, Biotin Conjugated?

When designing immunoassays using GST Antibody, Biotin Conjugated, researchers can implement two established methodological approaches:

Bridged Avidin–Biotin (BRAB) Method:

• Immobilization phase:

  • Coat solid phase (e.g., microplate) with primary capture antibody against your protein of interest

  • Block non-specific binding sites with appropriate blocking buffer

  • Add sample containing GST-tagged protein

• Detection phase:

  • Add biotin-conjugated GST antibody to form a "sandwich" with the captured protein

  • Wash thoroughly to remove unbound antibody

  • Add unlabeled (strept)avidin which binds to the biotin on the antibody

  • Add biotin-labeled enzyme (e.g., HRP or alkaline phosphatase)

  • Wash to remove unbound components

  • Add appropriate substrate for the enzyme and measure signal

Labeled Avidin–Biotin (LAB) Method:

• Immobilization phase:

  • Follow the same procedure as BRAB for immobilizing the target protein

  • Add biotin-conjugated GST antibody to form a "sandwich"

• Detection phase:

  • Add enzyme-labeled (strept)avidin (pre-conjugated) directly to the complex

  • Wash to remove unbound components

  • Add appropriate substrate and measure signal

The LAB method offers fewer steps and potentially higher sensitivity, while the BRAB method provides greater signal amplification potential. The choice between methods should be based on specific experimental requirements for sensitivity, specificity, and available reagents.

What are the recommended quality control procedures for experiments using GST Antibody, Biotin Conjugated?

Implementing rigorous quality control procedures is essential for generating reliable data with GST Antibody, Biotin Conjugated:

• Pre-experimental validation:

  • Perform titration experiments to determine optimal antibody concentration

  • Include appropriate positive and negative controls

  • Validate lot-to-lot consistency using standard samples

• Specificity controls:

  • Include samples containing known concentrations of GST from Schistosoma japonicum

  • Test cross-reactivity with GST from other species if relevant to your research

  • Use GST-negative samples to confirm absence of non-specific binding

• Biotin interference assessment:

  • Include samples with added biotin at various concentrations to evaluate potential interference

  • Consider using non-biotin detection systems in parallel for comparison

• Reproducibility measures:

  • Run technical replicates to assess intra-assay variation

  • Repeat experiments on different days to evaluate inter-assay variation

  • Document lot numbers, storage conditions, and preparation methods

• Data validation:

  • Analyze standard curves for appropriate dynamic range and linearity

  • Apply appropriate statistical methods to evaluate significance of results

  • Compare results with alternative detection methods when possible

These quality control procedures help ensure experimental rigor and enhance confidence in research findings when working with GST Antibody, Biotin Conjugated.

How can I mitigate biotin interference in immunoassays using GST Antibody, Biotin Conjugated?

Biotin interference can significantly impact the reliability of immunoassays utilizing GST Antibody, Biotin Conjugated. Researchers can implement several strategies to mitigate this interference:

• Sample pre-treatment:

  • Implement biotin-depletion steps using streptavidin-coated microparticles to remove excess free biotin

  • Dilute samples when appropriate to reduce biotin concentration below interference thresholds

  • Document known biotin supplementation in experimental subjects

• Assay design modifications:

  • Consider alternative immobilization systems when working with samples likely to contain high biotin levels

  • Increase the concentration of streptavidin in the assay to overcome competitive inhibition

  • Modify incubation times to allow for more complete binding in the presence of interfering biotin

• Validation approaches:

  • Compare results using both biotin-dependent and biotin-independent detection methods

  • Create standard curves with and without added biotin to establish correction factors

  • Perform spike-and-recovery experiments with known biotin concentrations

• Alternative detection strategies:

  • Consider direct enzyme conjugation to the primary antibody instead of using the biotin-(strept)avidin system

  • Explore other high-affinity systems such as His-tag or FLAG-tag systems (though with awareness of their lower binding affinities)

Implementing these strategies can help maintain assay integrity when working with samples potentially containing interfering levels of biotin.

What are common troubleshooting steps for weak or inconsistent signals when using GST Antibody, Biotin Conjugated?

When encountering weak or inconsistent signals in experiments using GST Antibody, Biotin Conjugated, consider the following systematic troubleshooting approach:

• Antibody integrity assessment:

  • Verify proper storage conditions were maintained

  • Check for visible precipitation or cloudiness in the antibody solution

  • Centrifuge the product if not completely clear after standing at room temperature

  • Consider testing a new lot if degradation is suspected

• Protocol optimization:

  • Titrate antibody concentration to determine optimal working dilution

  • Extend incubation times to allow more complete binding

  • Optimize washing steps to reduce background while preserving specific signal

  • Adjust blocking conditions to improve signal-to-noise ratio

• Detection system evaluation:

  • Ensure streptavidin conjugate is functional and properly stored

  • Verify substrate freshness and activity

  • Consider more sensitive detection methods if signal strength is insufficient

  • Test alternative streptavidin conjugates (e.g., different enzymes or fluorophores)

• Target accessibility issues:

  • Modify sample preparation methods to improve epitope exposure

  • Adjust buffer conditions (pH, salt concentration, detergents)

  • Consider gentler fixation methods if applicable

• Experimental controls:

  • Include positive controls with known GST concentrations

  • Use a different detection antibody against the same target as comparison

  • Perform parallel experiments with non-biotinylated antibodies

Systematic application of these troubleshooting steps can help identify and resolve issues leading to suboptimal performance of GST Antibody, Biotin Conjugated in experimental systems.

How can I optimize GST Antibody, Biotin Conjugated for specific applications?

Optimizing GST Antibody, Biotin Conjugated for specific research applications requires tailored approaches based on the particular technique being employed:

ELISA Optimization:

• Blocking optimization:

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

  • Optimize blocking time and temperature

  • Consider specialized blocking buffers for reducing non-specific binding

• Antibody concentration:

  • Perform checkerboard titration to determine optimal concentrations

  • Balance sensitivity needs with reagent conservation

  • Consider signal amplification systems for low-abundance targets

• Incubation parameters:

  • Compare room temperature vs. 4°C incubations

  • Test different incubation times (1-24 hours)

  • Evaluate static vs. shaking conditions

Immunofluorescence Optimization:

• Fixation methods:

  • Compare different fixatives (paraformaldehyde, methanol, acetone)

  • Optimize fixation time and temperature

  • Consider antigen retrieval methods if necessary

• Permeabilization:

  • Test different detergents (Triton X-100, Tween-20, saponin)

  • Optimize detergent concentration and exposure time

  • Balance membrane permeabilization with epitope preservation

• Detection enhancement:

  • Evaluate different streptavidin-fluorophore conjugates

  • Consider tyramide signal amplification for weak signals

  • Optimize mounting media to preserve fluorescence

Western Blotting Optimization:

• Transfer conditions:

  • Optimize transfer time and voltage

  • Consider different membrane types (PVDF vs. nitrocellulose)

  • Evaluate wet vs. semi-dry transfer methods

• Blocking and washing:

  • Test milk vs. BSA-based blocking buffers

  • Optimize washing buffer composition and washing times

  • Consider specialized low-background detection systems

• Detection parameters:

  • Compare different streptavidin-enzyme conjugates

  • Optimize substrate exposure time

  • Consider chemiluminescence vs. fluorescence detection

For all applications, empirical determination of optimal conditions through systematic testing is essential for achieving the best results with GST Antibody, Biotin Conjugated .