ST5 Antibody, Biotin conjugated

What is the mechanism behind biotin-streptavidin interaction in ST5 antibody applications?

The biotin-streptavidin interaction utilized in the ST5 antibody system is one of the strongest non-covalent interactions found in nature, with an affinity constant (KD) of 10^-14 to 10^-15. This is approximately 10^3 to 10^6 times higher than typical antigen-antibody interactions . This exceptionally high affinity allows for robust and stable detection systems in immunoassays.

The interaction mechanism involves:

• Biotin molecules conjugated to the ST5 antibody

• Streptavidin molecules that can bind to these biotin tags with extraordinary specificity

• Formation of a stable complex that resists harsh conditions including temperature extremes, pH variation, and proteolytic enzymes

This system provides significant advantages for ST5 protein detection, including:

• Signal amplification for detecting low concentrations of ST5 protein

• Decreased number of steps required for measurement

• Rapid quantitation of analyte

• Superior stability against manipulation and harsh reagents

How does biotin conjugation affect the binding properties of the ST5 antibody?

Biotin conjugation to the ST5 antibody is designed to preserve the antibody's natural binding properties while adding functionality. The biotin molecule is relatively small (240 Da) with a flexible valeric side chain, making it well-suited for protein labeling without significantly altering the antibody's structure or function .

Research indicates that properly executed biotin conjugation:

• Maintains the antibody's specificity for the ST5 target

• Preserves the binding affinity to the target antigen

• Adds the functionality of streptavidin binding

• Allows for indirect interaction between biomolecules while preserving natural binding properties

The ST5 antibody is specifically designed for human reactivity, targeting recombinant Human Suppression of tumorigenicity 5 protein (amino acids 3-280) . The biotin conjugation process is optimized to maintain the antibody's functional characteristics while enabling detection through the biotin-streptavidin system.

What are the optimal storage and handling conditions for ST5 Antibody, Biotin conjugated to maintain functionality?

Proper storage and handling are critical for maintaining the functionality of the ST5 Antibody, Biotin conjugated:

Storage recommendations:

• Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles that can degrade antibody activity

• The antibody is supplied in a buffer containing 50% Glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative

Handling guidelines:

• Allow the antibody to equilibrate to room temperature before opening

• Aliquot the antibody into smaller volumes upon first thaw to minimize freeze-thaw cycles

• When diluting for experiments, use buffers of similar composition to the storage buffer

• Work in clean, nuclease-free environments to prevent contamination

Following these storage and handling recommendations will help maintain the >95% purity and functionality of the Protein G purified antibody for the intended experimental applications .

What is the recommended protocol for using ST5 Antibody, Biotin conjugated in ELISA assays?

For optimal results in ELISA applications using ST5 Antibody, Biotin conjugated, the following protocol is recommended:

Materials needed:

• ST5 Antibody, Biotin conjugated (50μl or 100μl, depending on experimental scale)

• Appropriate blocking buffer (typically containing 1-5% BSA in PBS)

• Wash buffer (PBS with 0.05% Tween-20)

• Streptavidin-HRP conjugate

• Appropriate substrate (TMB, ABTS, etc.)

• 96-well microtiter plates

• Target antigen (recombinant ST5 protein)

Protocol:

• Coating: Coat wells with target protein or capture antibody (1-10 μg/ml) in coating buffer overnight at 4°C

• Blocking: Block non-specific binding sites with blocking buffer for 1-2 hours at room temperature

• Primary incubation: Apply samples containing ST5 protein and incubate for 1-2 hours at room temperature

• Secondary incubation: Dilute ST5 Antibody, Biotin conjugated at 1:500-1:1000 in blocking buffer and incubate for 1-2 hours at room temperature

• Detection: Add streptavidin-HRP (typically 1:1000-1:5000 dilution) and incubate for 30-60 minutes

• Substrate reaction: Add appropriate substrate and measure the signal

Optimization considerations:

• The antibody has been validated for ELISA applications with human ST5 protein

• Different detection systems (colorimetric, chemiluminescent, fluorescent) can be used with the biotin-streptavidin system

• The extremely high affinity of biotin-streptavidin (KD = 10^-14-10^-15) provides excellent sensitivity compared to other detection systems

How can I validate the specificity of ST5 Antibody, Biotin conjugated in my experimental system?

Validating antibody specificity is crucial for obtaining reliable research results. For ST5 Antibody, Biotin conjugated, consider the following validation approaches:

Positive controls:

• Use recombinant human ST5 protein (amino acids 3-280), which corresponds to the immunogen used to generate the antibody

• Include cell lines known to express ST5/DENND2B at detectable levels

Negative controls:

• Include samples from species other than human (the antibody is specifically reactive to human ST5)

• Use cell lines with confirmed absence or knockdown of ST5 expression

• Include isotype control antibodies (rabbit IgG biotin-conjugated) to assess non-specific binding

Blocking experiments:

• Pre-incubate the antibody with recombinant ST5 protein before application to demonstrate specificity

• Compare detection signal with and without blocking to confirm specific binding

Western blot validation:

• Although the product is primarily recommended for ELISA, Western blot can be used for validation

• Use the recommended dilution of 1:300-1:5000 for Western blot applications

• Confirm band size corresponds to expected molecular weight of ST5 protein

Cross-reactivity assessment:

• Test against closely related DENN domain-containing proteins to ensure specificity

• Examine potential cross-reactivity with other human proteins containing similar epitopes

How can I address high background signal issues when using ST5 Antibody, Biotin conjugated?

High background signal is a common challenge when working with biotin-conjugated antibodies. Several strategies can mitigate this issue:

Common causes and solutions:

Specific considerations for ST5 Antibody:

• The Suppression of tumorigenicity 5 protein is involved in signal transduction pathways , so certain cell types may have higher endogenous expression

• The biotin-(strept)avidin system can be vulnerable to interference from high levels of supplemental biotin

• Consider using BSA that is certified biotin-free in all buffers

• Include additional wash steps with high salt buffer (up to 500mM NaCl) to reduce non-specific interactions

What are the potential sources of false positive or false negative results when using ST5 Antibody, Biotin conjugated?

Understanding potential sources of erroneous results is crucial for accurate data interpretation:

False positive sources:

• Biotin interference: High levels of supplemental biotin in samples can create false positive or negative results depending on the assay format

• Endogenous biotin: Human samples may contain varying levels of endogenous biotin

• Cross-reactivity: The polyclonal nature of the antibody may result in binding to proteins with similar epitopes

• Hook effect: Extremely high concentrations of target can paradoxically decrease signal in sandwich assays

• Non-specific binding: Insufficient washing or blocking can lead to non-specific signal

False negative sources:

• Epitope masking: Protein interactions or modifications may mask the antibody binding site

• Degraded antibody: Improper storage leading to loss of antibody activity

• Suboptimal conditions: pH, salt concentration, or detergents that inhibit antibody-antigen interaction

• Matrix effects: Components in complex samples may interfere with binding

• Biotin-streptavidin inhibitors: Presence of compounds that disrupt biotin-streptavidin interaction

Mitigation strategies:

• Include appropriate positive and negative controls in every experiment

• Verify results using an alternative detection method or antibody

• Titrate antibody concentration to determine optimal signal-to-noise ratio

• Consider using a different antibody (non-biotin conjugated) for confirmation of results

• For complex samples, perform spike-and-recovery experiments to assess matrix effects

How can ST5 Antibody, Biotin conjugated be utilized in protein-protein interaction studies?

The biotin-conjugated ST5 antibody offers versatile applications for studying protein-protein interactions:

Co-immunoprecipitation (Co-IP) approach:

• Capture ST5 protein complexes using the biotin-conjugated antibody bound to streptavidin-coated beads

• Wash complexes under various stringency conditions to maintain specific interactions

• Elute and analyze binding partners through mass spectrometry or Western blotting

• The high affinity of the biotin-streptavidin interaction (KD = 10^-14-10^-15) provides stable complex isolation

Proximity labeling techniques:

• Use the biotin-conjugated antibody to identify proteins in close proximity to ST5

• Combine with crosslinking approaches to capture transient interactions

• Leverage the high stability of biotin-(strept)avidin system against manipulation, proteolytic enzymes, temperature and pH extremes

Pull-down assays:

• Immobilize the biotin-conjugated antibody on streptavidin-coated surfaces

• Capture ST5 protein from cell lysates

• Identify interacting partners through various analytical methods

• The biotin-streptavidin system is particularly useful for isolating and amplifying weak signals

What advanced experimental designs can incorporate ST5 Antibody, Biotin conjugated for studying signal transduction pathways?

ST5 protein is categorized under signal transduction research areas , making the biotin-conjugated antibody valuable for advanced pathway studies:

Multiplex immunoassay design:

• Combine the ST5 antibody with antibodies against other signaling proteins

• Use different detection systems (varying fluorophores, quantum dots) coupled to streptavidin

• Simultaneously detect multiple pathway components

• The biotin-streptavidin system offers amplification of weak signals, enabling detection of low-abundance signaling intermediates

Temporal signaling dynamics:

• Use the antibody to capture ST5 protein at different time points after stimulus

• Analyze phosphorylation states and complex formation

• Develop kinetic models of signaling through the DENN domain-containing protein pathway

• The high stability of the biotin-(strept)avidin interaction provides reliable quantitation across different experimental conditions

Spatial organization studies:

• Combine with microscopy techniques using streptavidin-conjugated fluorophores

• Visualize subcellular localization of ST5 protein

• Track movement during signal transduction events

• The small size of biotin (240 Da) minimizes interference with protein trafficking

How can modified triphosphate bridge biotinylation techniques be combined with ST5 Antibody, Biotin conjugated for RNA-protein interaction studies?

Advanced RNA-protein interaction studies can leverage both the biotin-conjugated ST5 antibody and biotinylated RNA techniques:

Integrated approach for RNA-protein complex identification:

• Use biotin-labelled cap analogues with modified triphosphate bridge for mRNA tagging, which increases mRNA stability while maintaining biological activity

• Simultaneously use ST5 Antibody, Biotin conjugated to track the protein

• Employ streptavidin-based pull-down to isolate complexes containing both components

• Analyze the composition and dynamics of ST5-RNA interactions

Methodological considerations:

• The mRNA cap structure can be tagged with biotin via modified cap analogues while maintaining cap-dependent translation efficiency

• These cap-biotinylated RNAs resist decapping by Dcp2, providing enhanced stability

• Combined with the biotin-conjugated ST5 antibody, this system allows for studying how ST5/DENND2B may interact with RNA processing machinery

Technical workflow:

• Generate cap-biotinylated RNA using biochemically optimized analogues

• Express ST5 protein in cellular systems

• Use ultraviolet-cross-linking to stabilize protein-RNA interactions

• Perform affinity capture using streptavidin-coated matrices

• Analyze complexes through proteomics and transcriptomics approaches

This combined approach leverages the advantages of biotin-(strept)avidin technology for both protein and RNA tagging, creating powerful tools for exploring the potential roles of ST5 in RNA biology.

How does the performance of biotin-conjugated ST5 antibody compare with other detection systems in terms of sensitivity and specificity?

The biotin-streptavidin detection system offers distinct advantages compared to other detection methods:

Comparative binding affinities:

Data adapted from reference

Performance advantages:

• The exceptionally high affinity of biotin-streptavidin provides superior sensitivity compared to most other detection systems

• The system offers robust stability against harsh conditions, enabling stringent washing steps that increase specificity

• Signal amplification capabilities allow detection of low-abundance ST5 protein in complex samples

• The small size of biotin minimizes steric hindrance that might affect antibody-antigen binding

Limitations compared to other systems:

• Potential interference from endogenous biotin in biological samples

• Less reversible than some other affinity systems, which can complicate elution steps

• May require specialized streptavidin reagents for optimal performance

What are the methodological approaches for dual-tagging systems incorporating ST5 Antibody, Biotin conjugated with other tagged molecules?

Dual-tagging systems provide powerful approaches for complex experimental designs:

Compatible tag combinations:

• ST5 Antibody, Biotin conjugated can be paired with antibodies utilizing different tags

• V5 tag proves effective in dual-tagging systems and can be combined with biotin tags to elucidate protein interactions and complex formations

• Other compatible tags include FLAG, HA, His, and GST tags

Methodological workflow for ST5-V5 dual tagging:

• Express recombinant ST5 protein with a V5 tag

• Use ST5 Antibody, Biotin conjugated to detect native ST5 protein

• Use Anti-V5 tag antibodies to detect the recombinant protein

• Compare binding patterns and interactions between native and recombinant forms

• Leverage the different detection systems to distinguish between various protein complexes

Advanced applications:

• Sequential immunoprecipitation to isolate specific subcomplexes

• Co-localization studies using differently labeled streptavidin (for biotin) and anti-V5 detection systems

• Competitive binding assays to identify binding sites and interaction domains

• Conformational studies comparing accessibility of different epitopes

This dual-tagging approach expands the analytical power of the ST5 Antibody, Biotin conjugated by allowing simultaneous tracking of multiple protein variants or complex components in sophisticated experimental designs.