SLC22A25 Antibody, HRP conjugated

What is SLC22A25 and what cellular functions does it perform?

SLC22A25 (Solute carrier family 22 member 25) is a membrane transport protein also known as UST6 or Organic anion transporter UST6. It functions as an integral component of the plasma membrane and exhibits inorganic anion exchanger activity and sodium-independent organic anion transmembrane transporter activity. This protein plays crucial roles in urate transmembrane transport, sodium-independent organic anion transport, and urate transport mechanisms . SLC22A25 is part of the broader SLC22 family, which is instrumental in the transport of endogenous compounds, drugs, and toxins across cell membranes, making it a significant player in drug metabolism and detoxification processes .

What are the key characteristics of SLC22A25 Antibody, HRP conjugated?

SLC22A25 Antibody, HRP conjugated is typically a rabbit-derived polyclonal antibody that specifically targets human SLC22A25 protein. The antibody is conjugated to Horseradish Peroxidase (HRP), an enzyme that facilitates colorimetric or chemiluminescent detection in various immunoassays. These antibodies are generally raised against a recombinant Human SLC22A25 protein fragment (amino acids 31-145) . They are supplied in a storage buffer containing preservatives such as 0.03% Proclin 300 and stabilizers like 50% Glycerol in PBS (pH 7.4) . The conjugation to HRP eliminates the need for secondary antibody incubation in detection protocols, thus streamlining experimental workflows.

What are the standard applications for SLC22A25 Antibody, HRP conjugated?

The primary validated application for SLC22A25 Antibody, HRP conjugated is ELISA (Enzyme-Linked Immunosorbent Assay) . While the unconjugated version of SLC22A25 antibody can be used for Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF), the HRP-conjugated version is optimized specifically for ELISA applications . For ELISA applications, the recommended dilution range is typically between 1:2000 and 1:10000, though researchers should optimize dilutions for their specific experimental conditions . The HRP conjugation provides direct enzymatic activity for colorimetric or chemiluminescent detection systems without requiring secondary antibody incubation.

What are the optimal storage conditions and handling practices for SLC22A25 Antibody, HRP conjugated?

For short-term storage (up to 2 weeks), SLC22A25 Antibody, HRP conjugated should be maintained at 2-8°C . For long-term storage, it is recommended to store the antibody at -20°C or -80°C in small aliquots to prevent freeze-thaw cycles, which can degrade antibody performance . When working with the antibody, allow it to equilibrate to room temperature before opening the vial to prevent condensation, which can introduce contaminants and accelerate degradation. It's advisable to centrifuge the vial briefly before opening to collect any solution that might be in the cap. The antibody is supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . Note that Proclin 300 is classified as a hazardous substance and should be handled by trained personnel only .

How should I validate the specificity of SLC22A25 Antibody, HRP conjugated in my experiments?

To validate the specificity of SLC22A25 Antibody, HRP conjugated, several complementary approaches should be employed:

• Positive Controls: Use cell lines or tissues known to express SLC22A25, such as HeLa cells, which have been validated for SLC22A25 expression .

• Negative Controls: Include samples from tissues or cell lines with low or no expression of SLC22A25.

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide (amino acids 31-145 of human SLC22A25) before application to your sample. This should significantly reduce or eliminate specific binding.

• Molecular Weight Verification: In Western blot applications (using the unconjugated version), verify that the detected band appears at the expected molecular weight of approximately 62 kDa for SLC22A25 .

• Cross-Validation: Compare results with alternative detection methods such as RT-PCR or RNA sequencing to confirm protein expression correlates with transcript levels.

• Knockdown/Knockout Validation: If possible, test the antibody on samples where SLC22A25 has been knocked down or knocked out to confirm absence of signal.

These validation steps are crucial for ensuring experimental rigor and reproducibility, particularly for research on less-characterized targets like SLC22A25.

What are the recommended dilution protocols for different experimental applications?

For ELISA applications using the HRP-conjugated SLC22A25 antibody, begin with a mid-range dilution (e.g., 1:5000) and perform a titration to determine the optimal concentration for your specific experimental conditions . The optimal dilution will provide a strong specific signal while minimizing background. When working with precious samples or when signal strength is uncertain, consider performing a preliminary dilution series (e.g., 1:1000, 1:3000, 1:9000) to identify the appropriate working range.

How does the HRP conjugation affect epitope binding and assay sensitivity compared to unconjugated SLC22A25 antibodies?

• Steric Hindrance: The attachment of the HRP enzyme (approximately 44 kDa) to the antibody may introduce steric hindrance that could affect epitope accessibility, particularly when the epitope is in a sterically constrained region of the protein. This effect is highly dependent on the specific conjugation chemistry and the location of conjugation sites relative to the antigen-binding domains.

• Sensitivity Trade-offs: While HRP conjugation eliminates the signal amplification step of secondary antibody binding, it provides direct enzymatic activity. Studies with similar antibody systems have shown that HRP-conjugated antibodies typically offer improved signal-to-noise ratios in ELISA applications compared to two-step detection systems, particularly when optimizing substrate incubation time .

• Binding Kinetics: The conjugation process may modify the antibody's binding kinetics, potentially affecting its association and dissociation rates with the target epitope (amino acids 31-145 of SLC22A25). Researchers should be aware that optimal incubation times may differ between conjugated and unconjugated versions of the same antibody.

• Assay Optimization: The HRP-conjugated SLC22A25 antibody typically requires different optimization parameters compared to the unconjugated version, including different working dilutions (generally higher dilutions for the conjugated version) and reduced incubation times due to the direct detection capability.

For critical comparative studies between different samples, maintaining consistent antibody lots and preparation methods is essential to minimize technical variability.

What are effective troubleshooting strategies for non-specific binding or weak signal when using SLC22A25 Antibody, HRP conjugated?

When troubleshooting weak signals specifically, consider that SLC22A25 is a membrane protein, which may require specialized extraction methods for optimal detection. For ELISA applications, ensure that the coating antigen concentration is sufficient and that the plate binding capacity is appropriate for the target. Additionally, verify that the HRP activity has not been compromised by improper storage or handling by testing the antibody with a known positive control .

For non-specific binding issues, implementing a more stringent washing protocol using PBS-T (PBS with 0.05-0.1% Tween-20) can help reduce background. Additionally, pre-absorbing the antibody with related proteins or using a more specific blocking agent tailored to your sample type can improve specificity.

How can SLC22A25 Antibody, HRP conjugated be used to investigate the role of SLC22A25 in drug metabolism and transport studies?

SLC22A25 Antibody, HRP conjugated can be strategically employed to investigate the role of SLC22A25 in drug metabolism and transport through several experimental approaches:

• Expression Correlation Studies: Using ELISA with the HRP-conjugated antibody, researchers can quantify SLC22A25 expression levels in various tissues or cell lines and correlate these with drug transport efficiency or metabolism rates. This approach helps identify tissues where SLC22A25 might play significant roles in drug disposition.

• Pharmacological Intervention Assays: After treating cells or tissues with drugs or compounds of interest, researchers can use the antibody to detect changes in SLC22A25 expression levels, potentially identifying drugs that modulate transporter expression as part of their mechanism of action or adverse effect profile.

• Transport Inhibition Studies: By combining the antibody detection with functional transport assays, researchers can correlate SLC22A25 protein levels with transport activity in the presence of various inhibitors, helping elucidate structure-function relationships and binding sites.

• Genetic Variation Impact Assessment: The antibody can be used to quantify SLC22A25 protein expression in samples from individuals with different genetic variants, allowing researchers to assess how polymorphisms affect protein expression levels, potentially explaining interindividual variability in drug response.

• Subcellular Localization Studies: Although primarily used for ELISA, related unconjugated antibodies against SLC22A25 can be used for immunofluorescence studies to examine changes in subcellular localization of the transporter in response to drug treatments or disease states.

These approaches collectively contribute to understanding how SLC22A25, as a member of the organic anion transporter family, participates in drug transport, metabolism, and elimination pathways .

What sample preparation techniques maximize SLC22A25 detection while preserving epitope integrity?

Optimal sample preparation for SLC22A25 detection requires careful consideration of its membrane protein nature:

• Membrane Protein Extraction: Since SLC22A25 is an integral component of the plasma membrane , use extraction buffers containing non-ionic detergents (such as 1% Triton X-100 or 0.5% NP-40) to efficiently solubilize the protein while preserving native conformation. For quantitative analyses, consider membrane fractionation protocols to enrich for plasma membrane proteins.

• Protease Inhibition: Include a comprehensive protease inhibitor cocktail in all extraction buffers to prevent degradation of SLC22A25, which could affect epitope integrity, particularly for the 31-145 amino acid region targeted by the antibody .

• Sample Handling: Process samples at 4°C whenever possible and minimize freeze-thaw cycles, as membrane proteins are particularly susceptible to denaturation and aggregation during temperature fluctuations.

• Denaturation Considerations: For applications requiring denatured proteins, use mild denaturing conditions (lower SDS concentrations, shorter heating times) to minimize epitope destruction while ensuring adequate protein denaturation.

• Phosphorylation Preservation: If phosphorylation status of SLC22A25 is relevant to your research, include phosphatase inhibitors (such as sodium orthovanadate, sodium fluoride, and β-glycerophosphate) in extraction buffers.

• Protein Quantification: Use detergent-compatible protein assays (such as BCA or Modified Lowry) to accurately quantify protein concentration in your samples, ensuring consistent loading across experiments.

For cell lysate preparation specifically, HeLa cells have been validated as expressing detectable levels of SLC22A25 and can serve as positive controls for your experiments .

How can SLC22A25 Antibody, HRP conjugated be incorporated into multiplex assay systems?

Incorporating SLC22A25 Antibody, HRP conjugated into multiplex assay systems requires careful consideration of detection systems and antibody compatibility:

• Sequential ELISA Approaches: For measuring multiple proteins in the same well, consider sequential detection protocols where the HRP-conjugated SLC22A25 antibody is used first, followed by thorough washing and HRP inactivation (using sodium azide or hydrogen peroxide quenching) before proceeding with subsequent antibodies with alternative enzyme conjugates (such as alkaline phosphatase).

• Spatially Resolved Multiplex ELISA: Implement microarray-based ELISA formats where different capture antibodies are spotted in discrete locations, allowing simultaneous detection of SLC22A25 and other proteins while avoiding signal interference.

• Substrate Selection: Choose HRP substrates with distinct spectral characteristics (such as TMB for colorimetric detection at 450 nm) that can be distinguished from other detection systems in your multiplex assay.

• Coordination with Other Conjugated Antibodies: When combining with other HRP-conjugated antibodies in multiplex systems, ensure that all antibodies have similar enzyme activity per molecule to provide consistent signal intensity across targets at equivalent protein concentrations.

• Validation Protocols: For any multiplex system, conduct comprehensive validation studies to ensure that the presence of multiple antibodies does not create unexpected cross-reactivity or interfere with SLC22A25 detection sensitivity.

For multiplexed analysis of transporter proteins including SLC22A25, consider compatibility with other solute carrier family antibodies to develop comprehensive transport protein profiling systems.

What are the considerations for using SLC22A25 Antibody, HRP conjugated in high-throughput screening applications?

Implementing SLC22A25 Antibody, HRP conjugated in high-throughput screening (HTS) requires optimization of several parameters to ensure reliability, reproducibility, and efficiency:

• Antibody Stability: For extended HTS campaigns, assess the stability of the HRP-conjugated antibody under automated handling conditions, including resistance to shear forces from automated pipetting and temperature fluctuations during plate handling.

• Protocol Miniaturization: Adapt standard ELISA protocols to 384-well or 1536-well formats, optimizing antibody concentrations and reaction volumes. Typical high-throughput ELISA formats may use 25-50% of the volumes recommended in standard protocols, which requires careful validation of antibody performance at these reduced volumes.

• Automation Compatibility: Ensure that the antibody performance is consistent under automated liquid handling parameters, particularly considering potential differences in dispensing pressures, aspiration rates, and mixing conditions compared to manual protocols.

• Signal Window Optimization: Determine the optimal substrate and development time that provides the largest signal window (difference between positive and negative controls) while maintaining linearity of response, critical for distinguishing hits in large compound libraries.

• Batch Effects Management: Implement robust quality control measures including multiple controls per plate and regular reference standard curves to mitigate batch effects across plates and screening days.

• Data Analysis Pipeline: Develop specialized data analysis workflows that account for the specific binding characteristics and signal generation kinetics of the HRP-conjugated SLC22A25 antibody, including appropriate normalization methods and hit selection criteria.

• Storage During Campaigns: For screening campaigns requiring multiple antibody aliquots, validate that performance remains consistent across aliquots and throughout the storage period under your specific laboratory conditions.

When using SLC22A25 Antibody, HRP conjugated in drug transporter screens, consider including known substrates and inhibitors of related organic anion transporters as reference compounds to benchmark assay performance and establish appropriate threshold criteria for hit identification.

How can SLC22A25 Antibody be used to investigate its role in physiological and pathological processes?

SLC22A25 Antibody provides a valuable tool for investigating this transporter's role in various biological contexts:

• Tissue Expression Profiling: Using ELISA with the HRP-conjugated antibody allows quantitative assessment of SLC22A25 protein expression across different tissue types, helping map the transporter's distribution and potential physiological roles. This is particularly valuable given SLC22A25's involvement in organic anion and urate transport .

• Disease State Correlation: By analyzing SLC22A25 expression in normal versus diseased tissues, researchers can identify potential associations with pathological conditions, particularly those involving disrupted metabolism or transport of endogenous compounds and xenobiotics.

• Renal Transport Studies: Given SLC22A25's urate transmembrane transporter activity , the antibody can be used to investigate its expression and function in kidney tissues, potentially elucidating its role in conditions like hyperuricemia, gout, or kidney stone formation.

• Drug-Induced Expression Changes: Monitoring SLC22A25 expression levels following exposure to various drugs or toxins can reveal potential regulatory mechanisms and adaptive responses in drug metabolism pathways.

• Genetic Variant Impact Assessment: By correlating SLC22A25 protein levels with specific genetic variants, researchers can investigate how polymorphisms affect expression and potentially contribute to interindividual variability in drug responses or disease susceptibility.

• Co-expression Analysis: Combining SLC22A25 detection with analysis of other transporters or metabolic enzymes can reveal coordinated expression patterns, suggesting functional networks involved in xenobiotic handling and endogenous compound homeostasis.

The ability to specifically detect and quantify SLC22A25 protein levels provides crucial complementary data to functional studies, helping bridge the gap between genetic information and physiological outcomes in transport biology.

What experimental approaches can determine the specific binding characteristics of SLC22A25 Antibody to its target epitope?

To comprehensively characterize the binding properties of SLC22A25 Antibody to its target epitope (amino acids 31-145) , several experimental approaches can be employed:

• Epitope Mapping: Perform fine epitope mapping using a series of overlapping peptides spanning the 31-145 region of SLC22A25 to identify the precise binding motif. This can be done through peptide arrays or ELISA with individual peptide fragments.

• Alanine Scanning Mutagenesis: Generate a series of single alanine substitutions throughout the epitope region and assess antibody binding to each mutant, identifying critical amino acid residues required for antibody recognition.

• Surface Plasmon Resonance (SPR): Determine binding kinetics parameters (kon, koff, KD) using SPR, comparing the HRP-conjugated version with the unconjugated antibody to assess how conjugation affects binding characteristics.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Use HDX-MS to identify regions of SLC22A25 that show protection from deuterium exchange when bound to the antibody, providing structural insights into the epitope-paratope interaction.

• Competitive Binding Assays: Assess whether the antibody competes with natural ligands or substrates of SLC22A25, which could indicate whether the epitope overlaps with functionally important regions of the transporter.

• Cross-reactivity Analysis: Test the antibody against closely related SLC22 family members to determine specificity boundaries and potential cross-reactivity patterns, which may reveal conserved epitope features within the transporter family.

These approaches collectively provide a comprehensive understanding of the antibody's binding characteristics, which is valuable for interpreting experimental results and designing future studies targeting SLC22A25.

What are emerging research applications for SLC22A25 Antibody in personalized medicine and pharmacogenomics?

SLC22A25 Antibody offers several promising applications in the evolving fields of personalized medicine and pharmacogenomics:

• Genotype-Phenotype Correlation Studies: The antibody enables researchers to quantify how genetic variants in SLC22A25 translate to protein expression levels, providing a critical link between genotype and functional phenotype. This information is valuable for interpreting pharmacogenomic data and predicting drug response variability.

• Biomarker Development: SLC22A25 expression patterns, detected using the antibody, may serve as biomarkers for predicting individual responses to drugs transported by this protein. ELISA-based quantification using the HRP-conjugated antibody could be developed into clinical diagnostic tools for personalized dosing strategies.

• Drug-Drug Interaction Profiling: By quantifying changes in SLC22A25 expression in response to various medications, researchers can identify potential drug-drug interactions mediated through transporter regulation, informing personalized polypharmacy management.

• Precision Therapeutic Selection: For drugs known to be substrates of SLC22A25, quantifying the transporter's expression in individual patients could guide therapeutic selection, directing patients toward medications that align with their transporter expression profile.

• Disease Susceptibility Assessment: Given SLC22A25's role in urate transport , expression profiling may help identify individuals at higher risk for conditions like gout or kidney stones, enabling preventive interventions.

• Ethnicity-Specific Medicine Development: By comparing SLC22A25 expression across different ethnic populations, researchers can identify population-specific patterns that might explain differential drug responses, contributing to more inclusive drug development.

These applications highlight how SLC22A25 Antibody contributes to the growing toolbox for personalized medicine approaches, particularly in understanding the molecular basis of variable drug responses and metabolic disease susceptibility related to transport protein function.