SLC22A10 Antibody

What is SLC22A10 and why is it important in research?

SLC22A10, also known as organic anion transporter 5 (OAT5), is a membrane protein encoded by the SLC22A10 gene in humans. It functions as an organic anion/dicarboxylic acid exchanger primarily localized in the proximal tubule straight segment (S3) of the kidney . This protein is significant in research because it interacts with numerous anionic drugs including bumetanide, furosemide, penicillin G, and various non-steroidal anti-inflammatory drugs . Its exclusive expression in kidney tissue makes it particularly valuable as a potential biomarker for kidney damage, as its urinary excretion may indicate early renal injury . The transporter's role in drug metabolism and excretion also makes it relevant for pharmacological and toxicological studies investigating drug interactions and elimination pathways.

What applications are SLC22A10 antibodies typically used for?

SLC22A10 antibodies are employed in multiple research applications with varying validated techniques:

When designing experiments, researchers should select antibodies specifically validated for their intended application. For instance, the recombinant monoclonal antibody 83237-4-PBS is particularly well-suited for developing ELISA assays as it forms part of a matched antibody pair (with 83237-5-PBS as the detection antibody) .

How should SLC22A10 antibodies be stored and handled to maintain optimal activity?

For optimal performance and longevity of SLC22A10 antibodies, researchers should follow specific storage and handling protocols:

Most commercial SLC22A10 antibodies are supplied in liquid form with specific storage buffers. For example, PACO64523 is provided in a preservative buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . Meanwhile, recombinant antibodies like 83237-4-PBS may be supplied in PBS only, without BSA or azide .

Storage temperatures vary by product, with some requiring -80°C storage (e.g., 83237-4-PBS) , while others may be stored at -20°C. Always follow manufacturer-specific guidelines to prevent degradation.

When working with these antibodies, minimize freeze-thaw cycles by aliquoting upon first use. Work in sterile conditions to prevent contamination, and use calibrated pipettes to ensure accurate dilutions for experimental applications. When preparing working dilutions, use recommended buffer systems that maintain protein stability and activity.

What are the critical factors affecting SLC22A10 antibody specificity, and how can cross-reactivity issues be addressed?

The specificity of SLC22A10 antibodies depends on several factors that researchers must carefully evaluate:

Immunogen design: SLC22A10 antibodies are generated against specific epitopes, such as the recombinant Human SLC22A10 protein segment (amino acids 37-145) used for PACO64523 . Understanding the exact immunogen helps predict potential cross-reactivity with related transporters in the SLC22 family.

Cross-reactivity assessment: Due to sequence homology among SLC22 family members, cross-reactivity testing is essential. Most commercial antibodies are validated specifically for human reactivity , but cross-species reactivity should be explicitly verified if working with animal models.

To address cross-reactivity concerns:

• Perform blocking peptide experiments using the specific immunogen peptide to confirm signal specificity

• Include appropriate negative controls (tissues or cells known not to express SLC22A10)

• Consider using knockout/knockdown validation approaches where possible

• When studying closely related transporters, use epitope mapping to select antibodies targeting unique regions

For highly sensitive applications, recombinant monoclonal antibodies like 83237-4-PBS may offer superior specificity compared to polyclonal alternatives, as they recognize a single epitope with consistent affinity across batches .

How can researchers optimize immunofluorescence protocols for detecting SLC22A10 in renal tissue sections?

Optimizing immunofluorescence (IF) protocols for SLC22A10 detection in kidney tissue requires attention to several technical considerations:

Fixation optimization: SLC22A10 is a multi-transmembrane protein with potential sensitivity to overfixation. While 4% formaldehyde fixation has been successful for cultured cells (as with SH-SY5Y cells using PACO64523) , renal tissue may benefit from shorter fixation times (4-8 hours) or alternative fixatives like PLP (periodate-lysine-paraformaldehyde) that better preserve membrane protein epitopes.

Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be empirically tested to determine optimal conditions for SLC22A10 detection in paraffin sections.

Blocking strategy: Given SLC22A10's localization in proximal tubules, a robust blocking protocol is essential:

• Block with 10% normal serum from the species of secondary antibody origin (e.g., goat serum)

• Include 0.1-0.3% Triton X-100 for membrane permeabilization

• Consider adding 1% BSA to reduce background

Antibody dilution and incubation: For primary antibodies like PACO64523, start with the recommended dilution range (1:100-1:500) and optimize through titration experiments. Extended incubation at 4°C (overnight) typically yields better signal-to-noise ratios compared to shorter incubations at room temperature.

Co-staining markers: Include proximal tubule markers (e.g., LTL, SGLT2) for co-localization studies to confirm the expected S3 segment expression pattern of SLC22A10 .

Detection system optimization: Use highly cross-adsorbed secondary antibodies to minimize background in multi-color immunofluorescence applications.

What strategies can resolve discrepancies between SLC22A10 protein expression results from different antibody-based detection methods?

When researchers encounter inconsistent results across different detection methods (e.g., Western blot vs. immunofluorescence vs. ELISA), systematic troubleshooting is required:

Method-specific optimization:

• For Western blotting: Evaluate protein extraction methods specifically optimized for membrane proteins (detergent selection is critical)

• For ELISA: Test different antibody pairs and blocking conditions

• For IF: Adjust fixation conditions to preserve the native epitope conformation

Epitope accessibility analysis: Different antibodies target different regions of SLC22A10. The protein contains 11 transmembrane domains , meaning some epitopes may be accessible only under specific denaturation conditions. Map which epitopes each antibody recognizes and analyze whether denaturation, reduction, or fixation might affect epitope exposure differently across methods.

Sample preparation consistency: Standardize lysis buffers, protein extraction protocols, and handling procedures across all experiments. For membrane proteins like SLC22A10, the detergent type and concentration critically affect solubilization and epitope preservation.

Validation with orthogonal methods: Complement antibody-based techniques with non-antibody methods such as:

• Mass spectrometry-based proteomics

• mRNA expression analysis (qPCR, RNA-seq)

• Functional transport assays

Reference standard inclusion: When possible, use recombinant SLC22A10 protein as a positive control across different detection methods to establish baseline detection sensitivity .

If discrepancies persist, consider testing multiple antibodies targeting different epitopes to build a comprehensive understanding of expression patterns.

How should researchers design experiments to investigate SLC22A10 antibody specificity in the context of multiple OAT family members?

Designing robust experiments to validate SLC22A10 antibody specificity requires systematic approaches addressing potential cross-reactivity with related OAT family transporters:

Expression system controls: Generate cell lines with defined expression profiles:

• HEK293 cells transfected with individual OAT family members (SLC22A10/OAT5, OAT1, OAT3, etc.)

• CRISPR/Cas9 knockout cell lines lacking endogenous SLC22A10 expression

• Cell lines with titrated expression levels via inducible promoters

Sequential immunodepletion: To identify potential cross-reactivity:

• Pre-absorb the antibody with recombinant proteins from related OAT family members

• Test the absorbed antibody against samples containing SLC22A10

• Compare signal reduction patterns to identify cross-reactivity

Epitope competition assay: Design peptides representing homologous regions across OAT family members and test their ability to compete with SLC22A10 detection.

Differential expression analysis: Compare antibody performance across tissue panels with known expression patterns of different OAT transporters. SLC22A10/OAT5 is primarily expressed in kidney , while other OAT family members have broader tissue distribution.

This comprehensive approach ensures that signals detected with SLC22A10 antibodies genuinely represent the target protein rather than related family members.

What are the critical considerations when using SLC22A10 antibodies for quantifying protein expression changes in renal pathology models?

When quantifying SLC22A10 expression changes in kidney disease models, researchers should address several methodological considerations:

Normalization strategy selection: For Western blot quantification, carefully select loading controls:

• Traditional housekeeping proteins (β-actin, GAPDH) may vary in expression during kidney injury

• Consider membrane protein-specific loading controls (Na⁺/K⁺-ATPase) or total protein normalization (Ponceau S, REVERT)

• For immunohistochemistry/IF, normalize SLC22A10 signal to proximal tubule markers to account for tubular injury or loss

Technical sampling considerations:

• Regional heterogeneity exists within kidneys; standardize sampling regions (cortex vs. medulla)

• For human biopsies or small animal samples, evaluate whether the sample contains the S3 segment where SLC22A10 is primarily expressed

• Document the proportion of proximal tubules present in each analyzed field

Quantification methodology:

• For IF/IHC: Distinguish between changes in expression intensity versus changes in the number of expressing tubules

• For Western blot: Use standard curves with recombinant SLC22A10 protein to establish the linear detection range

• For ELISA: Validate parallelism between standard curves and biological samples to ensure accurate quantification

Timing considerations: SLC22A10 expression may change dynamically during disease progression. Design time-course studies to capture early transient changes versus chronic adaptations in protein expression.

Functional correlation: Complement expression data with functional transport assays to determine whether changed protein levels correlate with altered transporter activity.

How can researchers effectively troubleshoot non-specific binding and background issues when using SLC22A10 antibodies in immunoassays?

Non-specific binding and high background are common challenges when working with antibodies against membrane transporters like SLC22A10. Systematic troubleshooting approaches include:

Western blot optimization:

• Increasing blocking stringency (5% BSA often superior to milk for membrane proteins)

• Testing gradient polyacrylamide gels to better resolve SLC22A10 (predicted MW ~60 kDa)

• Optimizing membrane transfer conditions for hydrophobic proteins (adding SDS to transfer buffer)

• Reducing primary antibody concentration while extending incubation time (overnight at 4°C)

• Testing different detergents in wash buffers (0.1% Tween-20 vs. 0.3% Triton X-100)

Immunofluorescence optimization:

• Implementing double blocking steps (serum followed by protein block)

• Using image acquisition settings determined from negative controls

• Employing tissue-specific autofluorescence quenching protocols

• Testing different permeabilization approaches (Triton X-100 vs. saponin)

ELISA troubleshooting:

• Comparing different plate blocking agents (BSA, casein, commercial blockers)

• Testing multiple wash protocols (varying stringency and number of washes)

• Evaluating the optimal antibody pair orientation for sandwich ELISA

• Using validated recombinant proteins as standard curves and positive controls

Universal approaches:

• Including isotype control antibodies at matching concentrations

• Testing antibodies on tissues/cells known to lack SLC22A10 expression

• Pre-absorbing antibodies with immunizing peptide as a specificity control

• Comparing multiple antibodies targeting different SLC22A10 epitopes

How can SLC22A10 antibodies be effectively incorporated into multiplex assays for studying transporter regulation in kidney injury models?

Developing multiplex assays to study SLC22A10 alongside other kidney transporters requires careful technical considerations:

Antibody compatibility assessment:
For multiplex IF/IHC:

• Select antibodies raised in different host species to avoid cross-reactivity

• Use highly cross-adsorbed secondary antibodies to minimize species cross-reactivity

• Establish single-staining controls before attempting multiplexing

• Consider using directly conjugated primary antibodies for complex multiplexing

For multiplex protein assays (Luminex/MSD):

• Validate the 83237-4-PBS/83237-5-PBS matched antibody pair in multiplex formats

• Test for cross-reactivity between detection antibodies and non-target capture antibodies

• Optimize capture antibody coupling density to microspheres

• Establish single-analyte standard curves before proceeding to multiplex format

Kidney injury biomarker panels:
SLC22A10 can be integrated into biomarker panels including:

Data normalization strategies:
For accurate comparisons across samples:

• Use total protein normalization (Bio-Rad TPN, REVERT)

• Include invariant reference proteins in multiplex panel

• Apply spatial normalization in image-based multiplexing

• Consider housekeeping gene stability in disease states

Validation requirements:

• Confirm multiplex assay results with single-plex measurements

• Validate against orthogonal methods (qPCR, MS proteomics)

• Perform spike-recovery experiments to assess matrix effects

What methodological approaches can distinguish between changes in SLC22A10 protein expression versus altered subcellular localization in experimental models?

Differentiating between changes in total SLC22A10 expression versus altered trafficking/localization requires specialized methodological approaches:

Subcellular fractionation protocols:

• Differential centrifugation to separate membrane fractions:

  • Low-speed pellet (nucleus, cell debris)

  • Medium-speed pellet (mitochondria, lysosomes)

  • High-speed pellet (plasma membrane, microsomes)

  • Ultracentrifugation pellet (vesicular fraction)

• Analyze SLC22A10 distribution across fractions using validated antibodies

• Confirm fraction purity using organelle-specific markers

Surface biotinylation assays:

• Label cell-surface proteins with membrane-impermeable biotinylation reagents

• Capture biotinylated proteins with streptavidin

• Quantify the ratio of surface-to-total SLC22A10 by Western blot

• Compare ratios across experimental conditions

Advanced microscopy approaches:

• Confocal microscopy with z-stack acquisition to assess apical versus basolateral localization

• Super-resolution microscopy (STORM, STED) for nanoscale localization

• FRAP (Fluorescence Recovery After Photobleaching) to assess membrane mobility

• Co-localization analysis with endosomal markers to track internalization

Protease protection assays:

• Treat intact cells with membrane-impermeable proteases

• Compare degradation patterns of SLC22A10 using domain-specific antibodies

• Analyze protected fragments to deduce topology and accessibility

When applying these methods, researchers should recognize that SLC22A10's normal localization is at the apical membrane of proximal tubule S3 segments . Changes in localization may indicate pathological trafficking defects or adaptive responses to injury.

How can researchers integrate SLC22A10 antibody-based studies with functional transport assays to comprehensively characterize this transporter in disease models?

Integrating structural (antibody-based) and functional studies provides comprehensive insights into SLC22A10 biology:

Correlation of expression and activity:

• Quantify SLC22A10 protein levels using validated antibodies (Western blot, ELISA)

• Measure transport activity of model substrates (e.g., estrone sulfate, ochratoxin A)

• Calculate activity-to-expression ratios to determine if transport efficiency changes

• Analyze whether post-translational modifications affect this relationship

Cell-based transport systems:

• Develop stable cell lines expressing SLC22A10 (HEK293, MDCK)

• Verify expression using antibody-based methods (IF, flow cytometry, Western blot)

• Measure uptake/efflux of fluorescent or radiolabeled substrates

• Correlate transport kinetics with protein expression levels

Ex vivo kidney slice models:

• Prepare precision-cut kidney slices from control or diseased tissue

• Immunostain sections to confirm SLC22A10 expression patterns

• Perform transport assays with fresh slices using model substrates

• Correlate regional transport activity with immunolocalization data

In vivo approaches:

• Administer probe drugs known to be SLC22A10 substrates

• Collect urine and plasma for pharmacokinetic analysis

• Harvest tissues for antibody-based expression analysis

• Correlate clearance parameters with protein expression/localization

Potential pitfalls and solutions:

This integrated approach allows researchers to determine whether observed pathological changes reflect alterations in expression, localization, or intrinsic activity of the SLC22A10 transporter.

How can advanced proteomics approaches complement antibody-based detection of SLC22A10 in biomarker discovery efforts?

Integrating antibody-based methods with advanced proteomics offers powerful approaches for SLC22A10 biomarker research:

Targeted MS-based proteomics:

• Develop SLC22A10-specific MRM (Multiple Reaction Monitoring) or PRM (Parallel Reaction Monitoring) assays

• Select signature peptides unique to SLC22A10 that avoid transmembrane domains

• Compare MS-based quantification with antibody-based measurements

• Use heavy-isotope labeled peptide standards for absolute quantification

Proximity-based proteomics:

• Employ antibody-guided proximity labeling (BioID, APEX) to identify SLC22A10 interactome

• Use validated SLC22A10 antibodies for immunoprecipitation prior to MS analysis

• Compare interactome changes during disease progression

• Identify novel regulatory proteins affecting SLC22A10 function

Post-translational modification mapping:

• Immunoprecipitate SLC22A10 using validated antibodies

• Analyze PTMs using LC-MS/MS (phosphorylation, ubiquitination, glycosylation)

• Correlate modifications with transporter function and cellular localization

• Develop modification-specific antibodies for key regulatory sites

Exosome proteomics:

• Isolate urinary exosomes containing membrane transporters

• Confirm SLC22A10 presence using antibody-based methods

• Perform comprehensive proteomics to identify co-regulated transporters

• Develop multi-marker panels including SLC22A10 for kidney injury detection

By combining the specificity of antibody-based approaches with the comprehensive capabilities of proteomics, researchers can develop more robust biomarker strategies and gain deeper mechanistic insights into SLC22A10 regulation in health and disease.

What considerations should researchers address when developing phospho-specific antibodies for studying SLC22A10 regulation?

Developing phospho-specific antibodies against SLC22A10 requires addressing several specialized considerations:

Phosphorylation site identification:

• Perform in silico analysis of potential phosphorylation sites using tools like NetPhos, PhosphoSitePlus

• Validate predicted sites using MS-based phosphoproteomics

• Prioritize sites based on evolutionary conservation and predicted functional impact

• Focus on sites in cytoplasmic domains accessible to kinases

Immunogen design strategy:

• Generate phosphopeptides containing the modified residue centrally positioned

• Include carrier proteins (KLH, BSA) for small phosphopeptides

• Consider dual-phosphorylation epitopes if regulation involves multiple sites

• Design corresponding non-phosphorylated peptides for negative selection

Validation requirements:

• Test antibody specificity against phosphorylated vs. non-phosphorylated peptides

• Validate using cells treated with phosphatase inhibitors vs. phosphatases

• Confirm specificity using CRISPR-modified cells with phospho-null mutations

• Demonstrate loss of signal following kinase inhibition or knockdown

Application optimization:

• Include phosphatase inhibitors in all sample preparation buffers

• Optimize fixation protocols to preserve phospho-epitopes

• Test signal enhancement methods (tyramide signal amplification)

• Validate detection in multiple applications (WB, IF, flow cytometry)

Researchers should recognize that membrane proteins like SLC22A10 present unique challenges for phospho-antibody development due to their hydrophobicity and complex tertiary structure. Focusing on cytoplasmic domains and regulatory regions most likely to undergo dynamic phosphorylation will increase the probability of successful antibody development.

What are the current limitations of available SLC22A10 antibodies, and how can researchers contribute to improving validation standards?

Current commercial SLC22A10 antibodies present several limitations that researchers should recognize:

Validation gaps:

• Limited validation across multiple applications (most antibodies validated only for 1-2 applications)

• Minimal cross-species validation despite evolutionary conservation

• Few antibodies validated against knockout/knockdown controls

• Limited epitope mapping information

Potential cross-reactivity issues:

• Sequence homology with other SLC22 family members may cause false positives

• Non-specific binding to other membrane proteins in certain applications

• Variable performance across tissue types and fixation conditions

Researchers can contribute to improved validation standards through:

Community-based validation initiatives:

• Contributing to antibody validation databases (Antibodypedia, ENCODE)

• Publishing detailed validation protocols as application notes or methods papers

• Sharing negative results to prevent repeated use of problematic antibodies

• Participating in multi-laboratory validation studies

Improved reporting practices:

• Documenting complete antibody information (catalog number, lot, dilution, incubation)

• Including all validation controls in publications

• Depositing full-resolution unprocessed images in public repositories

• Specifying exact immunogen sequences when known

Technical innovation:

• Developing SLC22A10 knockout cell lines as definitive negative controls

• Creating epitope-tagged SLC22A10 expression systems as positive controls

• Establishing CRISPR-engineered cell lines with modified epitopes

• Contributing to recombinant antibody development initiatives

What resources and protocols should researchers consult when designing comprehensive studies of SLC22A10 in kidney physiology and pathophysiology?

Researchers investigating SLC22A10 should utilize these resources and protocols:

Database resources:

• UniProt (Q63ZE4) for protein sequence and annotation

• Human Protein Atlas for tissue expression patterns

• NCBI Gene (387775) for genomic information and literature

• PharmGKB for pharmacogenomic associations

Experimental protocols:

• Optimized immunostaining protocols for kidney tissue:

  • 4% PFA fixation for 24 hours

  • Antigen retrieval in citrate buffer (pH 6.0)

  • Blocking with 10% normal goat serum

  • Primary antibody incubation at 4°C overnight

  • Detection with fluorophore-conjugated secondary antibodies

• Membrane protein extraction for Western blotting:

  • Specialized detergent-based extraction buffers (RIPA with 1% NP-40)

  • Avoiding boiling steps that may cause aggregation

  • Using gradient gels for optimal resolution

• Functional transport assays:

  • Fluorescent substrate uptake measurements

  • Radiolabeled substrate flux studies

  • Transport kinetics analysis protocols

Disease model resources:

• Established AKI and CKD rodent models with characterized SLC22A10 regulation

• Biobanks of human kidney disease samples with associated clinical data

• Patient-derived kidney organoids for translational studies

Collaborative networks:

• International Transporter Consortium (ITC) resources

• Kidney Precision Medicine Project (KPMP) protocols

• Human Kidney Tissue Atlas collaborative network