SLC16A2 Antibody

What is SLC16A2 and why is it significant for research?

SLC16A2 (Solute Carrier Family 16 Member 2) is an integral membrane protein that functions as a highly specific thyroid hormone transporter. In humans, the canonical protein consists of 539 amino acid residues with a molecular mass of approximately 59.5 kDa, though it may appear around 70 kDa in experimental conditions due to post-translational modifications . It belongs to the Monocarboxylate porter (TC 2.A.1.13) protein family and is primarily involved in metabolic processes, specifically the cellular uptake of thyroxine (T4), triiodothyronine (T3), reverse triiodothyronine (rT3), and diidothyronine . Its significance lies in its role in thyroid hormone homeostasis, with mutations in the SLC16A2 gene directly linked to Allan-Herndon-Dudley Syndrome, a rare X-linked intellectual disability disorder characterized by severe psychomotor retardation and abnormal thyroid hormone levels .

What alternative names and synonyms should researchers be aware of when searching for SLC16A2 antibodies?

When searching literature or product catalogs for SLC16A2 antibodies, researchers should be aware of several synonyms commonly used to describe this protein. These include MCT8 (Monocarboxylate transporter 8), MCT7 (Monocarboxylate transporter 7), XPCT (X-linked PEST-containing transporter), AHDS, MRX22, DXS128, and DXS128E . Among these, MCT8 is the most frequently used alternative designation in scientific literature. Database identifiers like gene ID 6567 and Swiss-Prot accession number P36021 are also valuable for ensuring accurate identification of the target protein when selecting antibodies . Awareness of these alternative designations is crucial for comprehensive literature searches and proper antibody selection, especially when comparing results across different studies that may use varied nomenclature.

What are the key considerations when selecting SLC16A2 antibodies for specific research applications?

When selecting SLC16A2 antibodies for research, several critical factors should be evaluated to ensure optimal experimental outcomes. First, application compatibility is essential – researchers should verify that the antibody has been validated for their specific application, whether Western blot, immunohistochemistry, immunofluorescence, or ELISA . The epitope targeted by the antibody is also crucial; some antibodies target the N-terminal region (amino acids 1-100), while others target middle regions (amino acids 101-200) . The choice of epitope can significantly impact detection of native versus denatured protein.

Species reactivity must be carefully considered – most commercial SLC16A2 antibodies react with human, mouse, and rat proteins, but cross-reactivity with other species varies . The clonality of the antibody affects experimental outcomes; polyclonal antibodies (often rabbit-derived) generally provide higher sensitivity for detecting low-abundance SLC16A2, while monoclonal antibodies offer greater specificity and consistency .

Additional factors include validation data (especially published citations), immunogen sequence, and the appropriate working dilution for specific applications (typically 1:300-5000 for Western blot and 1:5000-10000 for ELISA) . For membrane proteins like SLC16A2, antibodies raised against unique regions rather than conserved transmembrane domains help distinguish between related family members.

What is the optimal protocol for detecting SLC16A2 by Western blot?

The optimal Western blot protocol for SLC16A2 detection requires careful consideration of its properties as a membrane protein. For sample preparation, researchers should extract proteins from tissues known to express SLC16A2 (liver, heart, kidney) or relevant cell lines (LO2, HT-29, 293T) . Protein extraction should include protease inhibitors, with membrane protein-specific extraction methods often yielding better results for transmembrane proteins like SLC16A2.

For gel electrophoresis, 8-10% SDS-PAGE gels provide optimal separation, as SLC16A2 has a calculated molecular weight of ~59.5 kDa but is typically observed at ~70 kDa due to post-translational modifications . Transfer to PVDF membranes is generally preferred for transmembrane proteins. Blocking should be performed with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

Primary antibody incubation should follow manufacturer's recommended dilutions, typically 1:300-5000 for Western blot applications . The optimal dilution for SLC16A2 antibodies like those described in the search results ranges from 1:500-2000 . Incubation should occur overnight at 4°C with gentle rocking. After thorough washing, an appropriate HRP-conjugated secondary antibody should be applied, followed by ECL detection.

Researchers should expect to observe bands at approximately 59.5-70 kDa, though the actual observed molecular weight may differ from the calculated weight due to glycosylation and other post-translational modifications . Positive controls should include liver, heart, or kidney tissues from the appropriate species, as these have been validated for SLC16A2 expression .

How can researchers validate the specificity of SLC16A2 antibodies?

Validating SLC16A2 antibody specificity requires a multi-faceted approach to ensure reliable experimental results. First, researchers should employ positive and negative tissue controls, testing antibodies on tissues known to express SLC16A2 (liver, heart, kidney) and those with minimal expression . Comparing staining patterns with published literature provides initial validation.

Peptide competition assays represent a powerful validation method – pre-incubating the antibody with excess immunizing peptide should eliminate or significantly reduce specific signals while leaving non-specific binding intact . For example, with antibodies targeting amino acids 1-100 or 101-200 of SLC16A2, the corresponding peptide sequences should be used for competition .

A multiple antibody approach provides additional validation by using different antibodies targeting distinct epitopes of SLC16A2. True signals should show consistent localization and detection patterns across different antibodies. For definitive validation, genetic approaches using SLC16A2 knockdown (siRNA/shRNA) or knockout (CRISPR/Cas9) samples serve as powerful negative controls .

Mass spectrometry validation can provide the highest confidence level – immunoprecipitation with the antibody followed by mass spectrometry analysis can confirm the presence of SLC16A2 peptides in the immunoprecipitate . This approach can also identify potential cross-reactive proteins. Finally, recombinant protein controls, including testing against purified SLC16A2 and related MCT proteins, help assess potential cross-reactivity with other family members .

What strategies help differentiate SLC16A2 from other closely related monocarboxylate transporters?

Differentiating SLC16A2 from other monocarboxylate transporters requires strategic approaches to ensure specificity. Antibody selection is critical – researchers should choose antibodies raised against unique regions of SLC16A2, particularly the N-terminal and C-terminal domains which typically have lower sequence homology with other MCT family members . Antibodies targeting conserved transmembrane domains are more likely to cross-react with related transporters.

Expression pattern analysis provides another differentiation strategy. SLC16A2 has a distinct tissue distribution profile, being highly expressed in liver and heart . Comparing detected protein expression with known tissue-specific patterns of various MCT family members can help confirm identity. Similarly, subcellular localization patterns may differ between transporters.

Functional differentiation is particularly valuable – SLC16A2 specifically transports thyroid hormones (T3, T4, rT3), whereas other MCTs primarily transport monocarboxylates like lactate and pyruvate . Correlating protein detection with functional transport assays can confirm identity. Additionally, molecular weight discrimination can help, as SLC16A2 (539 aa, ~59.5-70 kDa observed) may differ from other MCTs like MCT1 (SLC16A1, ~43 kDa) .

For definitive differentiation, advanced approaches include mass spectrometry of immunoprecipitated proteins to identify unique peptides, CRISPR/Cas9 knockout controls, and molecular techniques like RT-PCR with specific primers targeting unique regions of SLC16A2 mRNA as a complementary approach to antibody-based detection .

What control samples are essential for SLC16A2 antibody experiments?

Robust experimental design for SLC16A2 research requires comprehensive controls to ensure valid interpretation of results. Positive tissue controls should include liver and heart samples, which demonstrate high endogenous expression of SLC16A2 . Mouse kidney, rat heart, and rat liver have been specifically validated as positive control tissues in previous research . For cell-based experiments, validated positive cell lines include LO2, HT-29, and 293T cells .

Negative controls are equally important and should include primary antibody omission, isotype control antibodies at matched concentrations, and pre-immune serum from the host species. Genetic controls such as SLC16A2 knockdown cells (siRNA/shRNA) or knockout models provide the most definitive negative controls .

Competitive inhibition controls add another validation layer. Pre-incubating the antibody with the immunizing peptide (corresponding to the epitope region) followed by parallel experiments with blocked and unblocked antibody helps confirm signal specificity . For antibodies targeting amino acids 1-100 or 101-200 of SLC16A2, the corresponding peptide sequences should be used .

Application-specific controls include molecular weight ladders and loading controls (β-actin, GAPDH) for Western blotting, known positive and negative tissue sections for immunohistochemistry, and non-specific IgG controls for immunoprecipitation. Including cross-species validation (testing on human, mouse, and rat samples) confirms species cross-reactivity claims by manufacturers and helps establish the conservation of epitope recognition .

How can SLC16A2 antibodies be utilized to investigate thyroid hormone transport mechanisms?

SLC16A2 antibodies enable sophisticated investigation of thyroid hormone transport mechanisms through multiple complementary approaches. Co-localization studies combine SLC16A2 antibodies with antibodies against thyroid hormone receptors or other transport proteins, using confocal microscopy to visualize potential interaction sites at the cellular level . This provides spatial information about the thyroid hormone transport machinery.

For functional studies, researchers can manipulate SLC16A2 expression through overexpression or knockdown approaches, using antibodies to confirm expression levels by Western blot or immunofluorescence . These protein level measurements can then be correlated with functional uptake of radiolabeled thyroid hormones (T3, T4, rT3), establishing quantitative relationships between protein levels and transport activity.

Structure-function studies represent another powerful application. By generating SLC16A2 mutants based on disease-associated variants, researchers can use antibodies to confirm expression and localization of mutant proteins . Comparing transport kinetics between wild-type and mutant transporters helps identify critical functional domains through targeted mutations and antibody detection.

Cell surface biotinylation techniques, where biotinylation reagents label cell surface proteins before isolation with streptavidin and detection of SLC16A2 using specific antibodies, allow quantification of membrane-localized versus intracellular SLC16A2 pools . This helps track SLC16A2 trafficking and surface expression under various conditions or in disease models. For in vivo studies, transgenic mouse models with tagged SLC16A2 can be validated using antibodies to confirm expression patterns before correlating with physiological markers of thyroid hormone action .

What approaches can be employed to study SLC16A2 protein-protein interactions?

Investigating SLC16A2 protein-protein interactions requires specialized techniques that accommodate its membrane protein characteristics. Co-immunoprecipitation (Co-IP) represents a foundational approach – using SLC16A2 antibodies to precipitate protein complexes from cell/tissue lysates, followed by identification of interacting partners by Western blot or mass spectrometry . For membrane proteins like SLC16A2, mild lysis buffers help preserve protein-protein interactions, and cross-linking prior to lysis can capture transient interactions.

Proximity Ligation Assay (PLA) offers an elegant in situ approach by combining SLC16A2 antibody with antibodies against potential interacting partners . Oligonucleotide-linked secondary antibodies generate signals only when proteins are in close proximity, allowing visualization of endogenous protein interactions with subcellular resolution.

For live-cell studies, techniques like Bimolecular Fluorescence Complementation (BiFC) or Förster Resonance Energy Transfer (FRET) require generating fusion constructs of SLC16A2 and potential partners with split fluorescent protein fragments or fluorescent tags . Expression and localization can be validated using SLC16A2 antibodies before performing interaction studies.

More specialized approaches include cross-linking mass spectrometry, where protein complexes are chemically cross-linked in intact cells before immunoprecipitation with SLC16A2 antibodies . Mass spectrometry identification of cross-linked peptides can provide structural information about interaction interfaces. For high-throughput screening, yeast two-hybrid or mammalian two-hybrid systems (preferably membrane-specific variants) can identify potential interactors, with hits subsequently validated using antibody-based methods in mammalian cells .

How can researchers investigate the role of SLC16A2 in Allan-Herndon-Dudley Syndrome?

Investigating SLC16A2's role in Allan-Herndon-Dudley Syndrome (AHDS) requires integrating molecular, cellular, and clinical approaches. Clinical sample analysis forms a foundation by performing immunohistochemistry on patient biopsies or available tissues, comparing SLC16A2 expression patterns between healthy and affected samples . Quantitative image analysis provides objective comparison of expression levels that can be correlated with clinical parameters.

Genetic variant characterization represents a central approach – expressing wild-type and disease-associated SLC16A2 variants in cell models allows researchers to use antibodies to assess total protein expression (Western blot), subcellular localization (immunofluorescence), and cell surface expression (surface biotinylation) . These protein characteristics can be correlated with functional thyroid hormone transport, linking molecular defects to disease mechanisms.

For more translational approaches, patient-derived cells or tissues can be analyzed for SLC16A2 expression and function . Creating patient-specific induced pluripotent stem cells (iPSCs) and differentiating them into neural cells allows investigation of how SLC16A2 mutations affect brain development. Antibodies play a crucial role in tracking abnormal cellular processing of mutant transporters in these models.

Therapeutic development represents an emerging application – researchers can screen compounds that may rescue mutant SLC16A2 trafficking or function, using antibodies to assess treatment effectiveness in restoring protein expression and localization . High-throughput cell-based assays can be developed for initial screening, with hits validated in patient-derived cells or animal models. These approaches collectively bridge basic research with clinical applications, potentially leading to new diagnostic or therapeutic strategies for AHDS .

What methods can be used to study post-translational modifications of SLC16A2?

Studying post-translational modifications (PTMs) of SLC16A2 requires specialized approaches that accommodate its membrane protein characteristics. For phosphorylation analysis, researchers can immunoprecipitate SLC16A2 using specific antibodies and probe with pan-phospho antibodies (anti-pSer, anti-pThr, anti-pTyr) . More definitively, mass spectrometry approaches can identify specific phosphorylation sites after phospho-enrichment of immunoprecipitated SLC16A2. Functional correlation can be established by creating phosphomimetic or phospho-dead mutants and comparing their localization and activity using antibody detection methods.

Glycosylation analysis is particularly relevant for SLC16A2, which may explain the discrepancy between its calculated (59.5 kDa) and observed (70 kDa) molecular weights . Enzymatic deglycosylation using PNGase F, Endo H, or O-glycosidase followed by Western blot with SLC16A2 antibodies can detect mobility shifts indicative of glycosylation . For detailed glycosylation site mapping, immunoprecipitated SLC16A2 can undergo glycopeptide enrichment followed by mass spectrometry identification of modified sites.

For ubiquitination and SUMOylation studies, co-immunoprecipitation approaches can be used – immunoprecipitating SLC16A2 and probing for ubiquitin/SUMO, or alternatively, precipitating ubiquitinated proteins and detecting SLC16A2 . For site identification, large amounts of immunoprecipitated SLC16A2 can be analyzed by mass spectrometry to identify modified lysine residues.

Integrated approaches examining PTM crosstalk provide valuable insights into SLC16A2 regulation. Researchers can study how one modification affects others by using phosphatase/glycosidase treatments followed by analysis of other PTMs . Stimulus-dependent modification changes (after treatment with hormones, drugs, or stress) can be correlated with functional outcomes, revealing regulatory mechanisms that may be relevant in normal physiology and disease states .

How can researchers address non-specific binding issues with SLC16A2 antibodies?

Non-specific binding is a common challenge with SLC16A2 antibodies that requires systematic troubleshooting. Optimization of blocking conditions represents a primary strategy – researchers should test different blocking agents (5% non-fat milk, 3-5% BSA, commercial blocking buffers) and extend blocking duration to 2 hours at room temperature or overnight at 4°C . Pre-blocking membranes or slides before primary antibody incubation can significantly reduce background.

Antibody dilution optimization is equally important. Researchers should perform titration series testing a range of dilutions (1:250, 1:500, 1:1000, 1:2000) to determine the optimal signal-to-noise ratio . The specific recommended dilutions for SLC16A2 antibodies vary by product and application – for example, 1:300-5000 for Western blot and 1:5000-10000 for ELISA with certain antibodies , or 1:500-2000 for Western blot with others .

Enhancing washing protocols often resolves persistent non-specific binding. Increasing washing stringency by adding higher concentrations of detergent (0.1-0.5% Tween-20), increasing the number of washes (5-6 times, 5-10 minutes each), or using higher salt concentration in wash buffer (up to 500 mM NaCl) can significantly reduce background .

Cross-adsorption techniques provide another effective approach. Pre-adsorbing the antibody with tissue/cell lysate from negative control samples or using tissue powder from the species of sample origin can remove antibodies that bind to common epitopes . Peptide competition experiments, where antibody is pre-incubated with excess immunizing peptide, help distinguish between specific and non-specific signals – true SLC16A2 bands will disappear while non-specific bands remain .

What factors contribute to variability in SLC16A2 molecular weight observations in Western blots?

The observed molecular weight of SLC16A2 in Western blots can vary significantly from its calculated value (59.5 kDa), with observations often around 70 kDa . This variability stems from several factors that researchers should consider when interpreting results. Post-translational modifications represent a primary factor – SLC16A2 undergoes glycosylation, which can add 5-15 kDa to its apparent molecular weight . This modification varies by cell/tissue type and physiological conditions. Researchers can verify glycosylation's contribution by treating samples with glycosidases before electrophoresis.

Protein processing events also contribute to molecular weight variability. SLC16A2 may have tissue-specific splice variants resulting in protein isoforms of different lengths . Additionally, proteolytic processing during sample preparation or as part of physiological regulation can generate fragments of varying sizes. Using antibodies targeting different regions of SLC16A2 can help identify such variants.

Technical variables significantly impact observed molecular weights. Sample preparation conditions, particularly the completeness of membrane protein denaturation, affect migration patterns . Overheating can cause aggregation or fragmentation, while insufficient denaturation can result in compact structures that migrate faster than expected. The gel percentage and type also affect resolution – gradient gels may show different apparent weights than fixed percentage gels.

Buffer and detergent effects further contribute to variability. Different extraction methods (RIPA buffer versus gentler NP-40 or Triton X-100 buffers) can affect protein conformation and associated detergents/lipids . SDS concentration in sample buffer impacts the degree of denaturation, particularly for membrane proteins like SLC16A2. Finally, species differences between human SLC16A2 (539 amino acids) and rodent orthologs may result in different migration patterns, highlighting the importance of species-appropriate positive controls .

How might advanced SLC16A2 antibody techniques contribute to therapeutic development for thyroid disorders?

Advanced SLC16A2 antibody techniques hold significant potential for therapeutic development in thyroid disorders through several innovative approaches. Patient-specific diagnostics represent an immediate application – SLC16A2 antibodies can help identify patients with altered transporter expression or localization, potentially indicating functional deficiencies even in the absence of mutations . Quantitative image analysis of biopsies or circulating biomarker development using SLC16A2 antibodies may facilitate personalized treatment approaches.

Drug discovery platforms leveraging SLC16A2 antibodies can accelerate therapeutic development. High-throughput screening assays using antibody-based detection of SLC16A2 trafficking or expression can identify compounds that rescue mutant transporter function or enhance wild-type activity . For Allan-Herndon-Dudley Syndrome, screens for small molecules that restore cell surface expression of mutant SLC16A2 could lead to mutation-specific therapies, with antibodies serving as crucial detection tools .

Targeted delivery systems represent another promising direction. Antibody-drug conjugates or nanoparticles decorated with SLC16A2-targeting moieties could potentially deliver therapeutic cargo directly to cells expressing the transporter . This approach might enable tissue-specific modulation of thyroid hormone signaling without systemic effects.

Mechanistic studies using advanced antibody techniques provide foundational knowledge for therapeutic development. Proximity ligation assays or FRET-based approaches can identify protein interaction partners that might serve as alternative therapeutic targets . Similarly, antibody-based studies of post-translational modifications might reveal regulatory pathways that could be pharmacologically modulated . These mechanistic insights, facilitated by increasingly sophisticated antibody applications, bridge basic science with translational advances, potentially yielding novel therapeutic approaches for both rare disorders like AHDS and more common thyroid conditions .

What emerging technologies might enhance the specificity and applications of SLC16A2 antibodies?

Emerging technologies promise to revolutionize SLC16A2 antibody research by enhancing specificity, expanding applications, and enabling new experimental paradigms. Single-cell antibody-based proteomics represents one frontier, allowing researchers to analyze SLC16A2 expression and modifications at the individual cell level within heterogeneous populations . Technologies like mass cytometry (CyTOF) or microfluidic antibody-based proteomics can reveal cell-type-specific SLC16A2 expression patterns that might be obscured in bulk tissue analyses.

Super-resolution microscopy techniques including STORM, PALM, and STED are transforming subcellular localization studies of membrane proteins like SLC16A2 . These approaches overcome the diffraction limit of conventional microscopy, enabling visualization of nanoscale distribution patterns and dynamic clustering of SLC16A2 within membrane microdomains. Combined with advances in antibody conjugation chemistry, these techniques permit multiplex imaging of SLC16A2 alongside interaction partners.

Recombinant antibody engineering is enhancing specificity through technologies like phage display to generate highly selective single-chain variable fragments (scFvs) or nanobodies against specific SLC16A2 epitopes or conformational states . These smaller antibody formats offer advantages for membrane protein targeting, including better access to sterically hindered epitopes and improved tissue penetration.

Spatially-resolved proteomics techniques such as Digital Spatial Profiling or Imaging Mass Cytometry combine antibody-based detection with precise spatial information, allowing researchers to map SLC16A2 distribution across complex tissues while preserving architectural context . This could reveal previously unappreciated regional variation in transporter expression relevant to thyroid hormone action. Finally, CRISPR-based tagging approaches enable endogenous labeling of SLC16A2, creating cellular models where antibodies can detect the native protein without overexpression artifacts, providing more physiologically relevant insights into transporter biology and potential therapeutic interventions .