SLC36A2 Antibody

What is SLC36A2 and what is its biological function?

SLC36A2 is a pH-dependent proton-coupled amino acid transporter belonging to the solute carrier family 36. In humans, the canonical protein has 483 amino acid residues with a molecular mass of 53.2 kDa. It primarily transports small, unbranched amino acids such as glycine, alanine, and proline. SLC36A2 is highly expressed in kidney and muscle tissues, where it contributes to amino acid homeostasis. The protein localizes to the endoplasmic reticulum and cell membrane, with up to three different isoforms reported. Functionally, SLC36A2 is involved in ion transport and has been associated with hyperglycinuria, a condition characterized by excessive glycine excretion in urine .

How does SLC36A2 differ from other members of the SLC36 family?

While both SLC36A2 and its paralog SLC36A1 are expressed in neurons, they demonstrate different subcellular localizations that suggest distinct physiological roles. SLC36A2 localizes primarily to the endoplasmic reticulum and recycling endosome, whereas SLC36A1 is expressed in the lysosome. This differential localization pattern indicates that SLC36A2 likely contributes to neuronal transport and sequestration of specific amino acids (glycine, alanine, and proline) in distinct cellular compartments. The specificity of SLC36A2 antibodies is critical in research, as they are typically designed not to cross-react with other members of the SLC36 protein family .

What are the known disease associations of SLC36A2?

Mutations in the SLC36A2 gene have been directly associated with iminoglycinuria and hyperglycinuria, metabolic disorders characterized by excessive urinary excretion of imino acids and glycine. These conditions reflect the protein's physiological role in amino acid reabsorption in the kidney. Understanding these disease associations provides valuable insights for researchers investigating renal physiology, amino acid metabolism disorders, and potential therapeutic targets for associated conditions .

What are the most effective applications for SLC36A2 antibodies in research?

SLC36A2 antibodies have been validated for multiple research applications, with ELISA and Western Blot being the most widely used and reliable techniques. Additionally, immunohistochemistry (IHC) and immunofluorescence (IF) are effective for tissue localization studies. For Western Blot applications, researchers typically observe a band at approximately 68 kDa (despite the calculated molecular weight of 53.2 kDa), likely due to post-translational modifications. When designing experiments, researchers should consider the specific antibody validation data and recommended working dilutions - typically 1-2 μg/ml for Western Blot applications .

How should I optimize antibody dilutions for different experimental techniques?

Optimization of antibody dilutions is essential for generating reliable results across different experimental techniques. For Western Blot analysis, a starting dilution of 1-2 μg/ml is recommended, while ELISA applications may require different concentrations. The optimal working concentration varies based on tissue type, protein expression levels, and specific experimental conditions. A systematic titration approach is advised, starting with the manufacturer's recommended dilution and adjusting as necessary based on signal-to-noise ratio. For experiments involving human stomach tissue lysate, successful detection has been achieved at 1 μg/ml concentration, which can serve as a reference point for similar tissues .

What are the optimal storage conditions for SLC36A2 antibodies?

SLC36A2 antibodies should be stored according to manufacturer recommendations to maintain their reactivity and specificity. Most commercial antibodies can be stored at 4°C for up to three months for ongoing experiments. For long-term storage, -20°C is recommended, where antibodies typically remain stable for up to one year. Antibodies are generally supplied in PBS containing 0.02% sodium azide as a preservative. Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and reduced antibody activity. Aliquoting antibodies before freezing is advised for antibodies that will be used in multiple experiments over time .

How can I reconstitute lyophilized SLC36A2 antibodies to maintain maximum activity?

While many commercial SLC36A2 antibodies are supplied in liquid form, some may be provided as lyophilized powder. For reconstitution, using sterile techniques and appropriate buffer systems is crucial. Typically, reconstitution in PBS (pH 7.4) or the manufacturer's recommended buffer is advised. Allow the antibody vial to warm to room temperature before opening to prevent condensation. Add the buffer slowly while gently rotating the vial to avoid protein denaturation. After reconstitution, allow the solution to sit at room temperature for 10-15 minutes before aliquoting and storing at the recommended temperature. Following reconstitution, validation of antibody activity through a test experiment is advisable before using in critical research applications .

How can I address non-specific binding in Western Blot experiments?

Non-specific binding is a common challenge when working with SLC36A2 antibodies in Western Blot applications. If observing multiple bands or high background, several methodological adjustments can improve specificity. First, increase the blocking time or concentration (5% BSA or milk in TBST is typically effective). Second, optimize antibody concentration - excessive antibody can lead to non-specific binding, so titrating down from the recommended concentration may help. Third, increase the number or duration of washing steps between antibody incubations. Fourth, include 0.1-0.5% Tween-20 in antibody dilution buffers to reduce hydrophobic interactions. Finally, pre-absorption of the antibody with the immunogen peptide can help identify which bands are specific to SLC36A2 versus non-specific interactions .

Why might I observe a discrepancy between the predicted molecular weight (53.2 kDa) and the observed molecular weight (68 kDa) for SLC36A2?

The discrepancy between predicted (53.2 kDa) and observed (68 kDa) molecular weights for SLC36A2 is a common phenomenon that can be attributed to several factors. Post-translational modifications, particularly glycosylation of the protein, can significantly increase the apparent molecular weight on SDS-PAGE. SLC36A2 contains potential N-glycosylation sites that, when modified, alter protein migration. Additionally, the hydrophobic nature of membrane proteins like SLC36A2 can affect SDS binding and result in anomalous migration patterns. The presence of splice variants (up to 3 isoforms have been reported) may also contribute to size differences. This observed discrepancy is consistent across multiple antibodies and sample types, suggesting it reflects the biological characteristics of the protein rather than artifacts of the detection method .

What strategies can improve immunohistochemical detection of SLC36A2 in fixed tissues?

Optimizing immunohistochemical detection of SLC36A2 requires attention to several methodological details. First, antigen retrieval methods should be carefully selected; for membrane proteins like SLC36A2, citrate buffer (pH 6.0) with heat-induced epitope retrieval often yields good results. Second, extended blocking (1-2 hours) with serum matching the secondary antibody host can reduce background. Third, overnight primary antibody incubation at 4°C at concentrations of 1-5 μg/ml typically improves specific signal detection. Fourth, for kidney tissues where SLC36A2 is highly expressed, reducing antibody concentration may be necessary to prevent oversaturation. Finally, amplification systems such as avidin-biotin complexes or tyramide signal amplification can enhance detection sensitivity for tissues with lower expression levels. Comparing results with multiple fixation methods (formalin, paraformaldehyde, and methanol) can help determine optimal conditions for epitope preservation .

How can SLC36A2 antibodies be utilized to investigate protein-protein interactions in transport complexes?

Investigating SLC36A2's protein interaction network requires sophisticated methodological approaches. Co-immunoprecipitation (Co-IP) with SLC36A2 antibodies can identify binding partners, particularly when performed under mild detergent conditions that preserve membrane protein complexes. For more stringent validation, proximity ligation assays (PLA) using SLC36A2 antibodies in combination with antibodies against suspected interaction partners can visualize protein interactions in situ with high specificity. Fluorescence resonance energy transfer (FRET) experiments using fluorophore-conjugated SLC36A2 antibodies can detect close-proximity interactions in live cells. When designing such experiments, researchers should be aware that the large size of antibodies may sterically hinder some interactions, so complementary approaches like cross-linking studies or yeast two-hybrid screening should be considered for comprehensive interaction mapping .

What considerations are important when using SLC36A2 antibodies in studies of neuronal amino acid transport?

When investigating neuronal SLC36A2 function, researchers must consider several technical and biological factors. First, the subcellular localization of SLC36A2 to the endoplasmic reticulum and recycling endosomes in neurons differs from its paralog SLC36A1 (localized to lysosomes), requiring high-resolution imaging techniques like confocal or super-resolution microscopy for accurate differentiation. Second, co-localization studies with organelle markers (e.g., calnexin for ER, Rab11 for recycling endosomes) are essential to confirm subcellular distribution. Third, functional transport studies should account for SLC36A2's preference for glycine, alanine, and proline versus other amino acids. Fourth, when studying disease models, researchers should consider that SLC36A2 mutations may affect protein localization rather than expression levels, necessitating careful immunofluorescence analysis rather than relying solely on Western Blot quantification. Finally, neuronal studies benefit from comparison between in vitro culture systems and in vivo tissue analyses to account for the complexity of the neuronal microenvironment .

How can I design experiments to investigate the role of SLC36A2 in disease models related to hyperglycinuria?

Designing experiments to investigate SLC36A2's role in hyperglycinuria requires a multi-faceted approach. First, genetically modified cell lines or animal models carrying disease-associated SLC36A2 mutations should be established using CRISPR/Cas9 or similar gene editing techniques. Antibody-based detection can then be used to assess how these mutations affect protein expression, localization, and stability. Functional transport assays measuring uptake of radiolabeled or fluorescently labeled amino acids (particularly glycine) can quantify the impact of mutations on transport activity. For in vivo studies, immunohistochemistry of kidney sections can evaluate SLC36A2 expression patterns in proximal tubules where reabsorption occurs. Correlation between SLC36A2 localization/function and physiological parameters (urinary amino acid levels, particularly glycine) provides crucial insights into pathophysiological mechanisms. Additionally, pharmacological interventions targeting pathways that regulate SLC36A2 activity can be evaluated as potential therapeutic approaches using these disease models .

How do different antibody conjugates affect the utility of SLC36A2 antibodies in various applications?

Different antibody conjugates expand the utility of SLC36A2 antibodies across various experimental platforms. Unconjugated antibodies offer maximum flexibility for customized detection strategies and are typically used with secondary antibody detection systems in Western Blot, immunohistochemistry, and immunofluorescence. FITC-conjugated SLC36A2 antibodies enable direct fluorescence detection, eliminating the need for secondary antibodies in flow cytometry and immunofluorescence, which reduces background and simplifies multiplexing with other markers. HRP-conjugated antibodies provide direct enzymatic detection for ELISA and Western Blot applications, enhancing sensitivity and reducing protocol steps. When selecting a conjugate, researchers should consider potential interference with protein function (particularly for studies of living cells), sensitivity requirements, spectral compatibility with other fluorophores in multiplex experiments, and whether quantitative analysis is needed (where consistent signal-to-noise ratios are essential) .

What are the recommended approaches for quantitative analysis of SLC36A2 expression using antibody-based methods?

Quantitative analysis of SLC36A2 expression requires standardized methodologies to ensure accuracy and reproducibility. For Western Blot quantification, normalization to housekeeping proteins (such as β-actin, GAPDH, or α-tubulin) using densitometry software is essential to account for loading variations. When comparing expression across different tissues, standard curves using recombinant SLC36A2 protein can provide absolute quantification. For immunohistochemistry, digital image analysis using specialized software that measures staining intensity and distribution provides semi-quantitative data. Flow cytometry offers quantitative single-cell analysis of SLC36A2 expression, particularly valuable for heterogeneous cell populations. ELISA provides the most precise quantification, especially when using a standard curve of recombinant protein. Regardless of method, technical replicates (minimum n=3) and biological replicates are necessary for statistical validity, and consistent antibody concentrations, exposure times, and image acquisition parameters must be maintained across compared samples .

How can I differentiate between SLC36A2 isoforms using antibody-based techniques?

Differentiating between the reported three isoforms of SLC36A2 requires strategic experimental design. Western Blot analysis using gradient gels (8-15% acrylamide) can improve resolution of closely sized isoforms. Selection of antibodies targeting epitopes in regions that differ between isoforms is crucial - the N-terminal region antibodies might detect specific isoforms differently than those targeting conserved regions. Isoform-specific PCR primers should be used alongside antibody detection to correlate protein and mRNA expression patterns. For tissues expressing multiple isoforms, 2D gel electrophoresis followed by Western Blot can separate isoforms based on both molecular weight and isoelectric point. Immunoprecipitation with one antibody followed by Western Blot with another can help validate isoform identity. Finally, mass spectrometry analysis of immunoprecipitated SLC36A2 provides definitive identification of specific isoforms based on unique peptide sequences, though this approach requires highly specific antibodies for the initial immunoprecipitation step .

How might SLC36A2 antibodies contribute to understanding the protein's role in neurological disorders beyond known associations?

While SLC36A2 has established associations with hyperglycinuria, its neuronal expression pattern suggests potential roles in neurological disorders that remain unexplored. SLC36A2 antibodies can facilitate research into these potential connections through several approaches. First, comparative immunohistochemical studies of brain tissues from patients with various neurological disorders versus healthy controls may reveal altered expression or localization patterns. Second, co-localization studies with neurotransmitter transporters and receptors could identify functional interactions relevant to neurological disease mechanisms. Third, investigating SLC36A2's role in glycine transport is particularly relevant given glycine's dual role as an inhibitory neurotransmitter and NMDA receptor co-agonist, potentially linking SLC36A2 to disorders involving glycinergic neurotransmission such as hyperekplexia, certain forms of epilepsy, and pain disorders. Fourth, SLC36A2 antibodies can be used to monitor protein expression changes in response to pharmacological interventions, providing insights into therapeutic mechanisms. This research direction represents an important frontier in understanding the broader implications of SLC36A2 beyond its established functions .