SLC6A13 Antibody, Biotin conjugated

What is SLC6A13 and what is its functional significance in neurobiology?

SLC6A13, also known as GAT-2 (GABA transporter 2), is a high-affinity gamma-aminobutyric acid (GABA) transporter that catalyzes sodium and chloride-dependent transport of GABA to terminate GABA-mediated synaptic activity. This protein is critical for maintaining proper neurotransmitter balance in the central nervous system. SLC6A13 terminates the biological actions of GABA through reuptake into cells, utilizing sodium and chloride concentration gradients. Research has also indicated that SLC6A13 may facilitate beta-alanine transport in addition to its primary role in GABA transport. SLC6A13 has been associated with several neurological conditions, including traumatic glaucoma and neovascular glaucoma, making it an important target for neurobiological research .

What is the molecular structure and biochemical properties of SLC6A13?

SLC6A13 is a transmembrane protein with a calculated molecular weight of approximately 68 kDa (specifically 68,009 Da) and comprises 602 amino acids. The protein functions as a membrane-embedded transporter with multiple transmembrane domains that facilitate the movement of GABA across cell membranes. Its structure includes binding sites for both sodium and chloride ions, which are essential co-transported ions for GABA uptake. The protein is encoded by the SLC6A13 gene (Gene ID: 6540), which has been mapped and characterized in humans (UniProtID: Q9NSD5) and is conserved across several mammalian species .

What are the distinguishing characteristics of biotin-conjugated SLC6A13 antibodies?

Biotin-conjugated SLC6A13 antibodies feature covalently attached biotin molecules that enable high-affinity binding to avidin or streptavidin conjugates in detection systems. This conjugation provides several advantages: (1) signal amplification due to the multiple biotin-binding sites on avidin/streptavidin; (2) increased detection sensitivity in complex biological samples; and (3) compatibility with various secondary detection methods. Commercially available biotin-conjugated SLC6A13 antibodies, such as the one from AFG Scientific (catalog #A70960), are typically generated using recombinant human SLC6A13 protein fragments (amino acids 1-40) as immunogens and are purified to >95% purity using Protein G affinity methods .

What applications are validated for SLC6A13 antibodies?

SLC6A13 antibodies have been validated for multiple experimental applications with varying levels of optimization required. The most commonly validated applications include ELISA (enzyme-linked immunosorbent assay), which is supported by all major commercial antibodies reviewed. Western blot (WB) applications have been validated for several antibodies, including those from Boster Bio (A07153) and CUSABIO (CSB-PA889104LA01HU), with recommended dilutions typically ranging from 1:500 to 1:2000 . Some antibodies have also been validated for immunohistochemistry (IHC) and immunofluorescence (IF) applications, although these require careful optimization of fixation and permeabilization protocols. The biotin-conjugated variants are particularly useful for ELISA applications where enhanced sensitivity is required .

How should sample preparation be optimized for SLC6A13 detection?

Optimal sample preparation for SLC6A13 detection depends on the tissue type and experimental application. For protein extraction from neural tissues, use of mild detergents such as 1% Triton X-100 or RIPA buffer supplemented with protease inhibitors is recommended to maintain the native conformation of the transporter protein. For fixed tissue sections, paraformaldehyde (4%) fixation is generally preferred over methanol fixation to preserve the membrane structure where SLC6A13 is localized. When using biotin-conjugated antibodies, it's crucial to block endogenous biotin in tissue samples (particularly in liver, kidney, and brain tissues) using streptavidin/biotin blocking kits prior to antibody incubation. For cell culture samples, gentle fixation protocols should be employed to avoid disrupting the membrane localization of the transporter .

What are the recommended dilution factors for SLC6A13 antibodies in different applications?

The optimal dilution factors for SLC6A13 antibodies vary by application, antibody source, and sample type. Based on manufacturer recommendations from multiple sources, the following dilution ranges should serve as starting points for optimization:

Researchers should always perform dilution series experiments when working with new antibody lots or tissue types to determine optimal signal-to-noise ratios .

How can non-specific binding be minimized when using biotin-conjugated SLC6A13 antibodies?

Non-specific binding is a common challenge when working with biotin-conjugated antibodies due to endogenous biotin present in many tissues. To minimize this issue, implement a comprehensive blocking strategy: (1) Block endogenous biotin using commercial avidin/biotin blocking kits before antibody incubation; (2) Increase the concentration of blocking agents (5-10% normal serum from the species of your secondary reagent); (3) Add 0.1-0.3% Triton X-100 to your blocking buffer to reduce hydrophobic interactions; (4) Consider using specialized blocking reagents containing non-fat dry milk or fish gelatin for particularly problematic samples; and (5) Perform negative controls using non-immune IgG of the same species and concentration as your primary antibody. When optimizing, gradually increase wash stringency using PBS-T (PBS with 0.05-0.1% Tween-20) and extend wash durations if background persists .

What storage conditions optimize the shelf-life of SLC6A13 antibodies?

Proper storage is critical for maintaining antibody performance over time. Most commercial SLC6A13 antibodies are supplied in a stabilizing buffer containing 50% glycerol and should be stored at -20°C for long-term preservation. These storage conditions typically ensure stability for at least one year post-shipment. For biotin-conjugated variants, which may be more sensitive to degradation, storage at -80°C may further extend shelf-life. Repeated freeze-thaw cycles significantly reduce antibody performance; therefore, preparing small working aliquots upon receipt is strongly recommended. For short-term storage and frequent use (within one month), refrigeration at 4°C is acceptable for most formulations. Always protect biotin-conjugated antibodies from prolonged light exposure, as this can affect the biotin moiety. The presence of preservatives like sodium azide (0.02-0.03%) or Proclin 300 (0.03%) in the storage buffer helps prevent microbial contamination but may interfere with certain applications like cell culture .

How can weak signals be enhanced when using SLC6A13 antibodies?

Weak signal detection is a common challenge that can be addressed through multiple optimization approaches. First, consider implementing signal amplification systems like tyramide signal amplification (TSA) which can increase detection sensitivity by up to 100-fold for biotin-conjugated antibodies. Second, optimize antigen retrieval methods—for formalin-fixed tissues, try citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) with heat-induced epitope retrieval. Third, reduce stringency of wash buffers by decreasing detergent concentration and shortening wash times. Fourth, extend primary antibody incubation time to overnight at 4°C to allow for greater epitope binding. Fifth, for Western blot applications, increase protein loading (50-100 µg/lane) and consider using PVDF membranes instead of nitrocellulose for better protein retention. Finally, for biotin-conjugated antibodies specifically, ensure you're using fresh streptavidin conjugates as they can lose activity over time. When these approaches are combined systematically, detection sensitivity can be significantly improved while maintaining specificity .

How can SLC6A13 antibodies be utilized in co-localization studies with other neurotransmitter transporters?

Co-localization studies examining SLC6A13 alongside other neurotransmitter transporters require careful experimental design to ensure specificity and minimize cross-reactivity. Begin by selecting antibodies raised in different host species to enable simultaneous detection (e.g., rabbit anti-SLC6A13 with mouse anti-GAT1). For fluorescence microscopy applications, implement multi-color immunofluorescence protocols using compatible fluorophores with minimal spectral overlap. When using biotin-conjugated SLC6A13 antibodies, pair them with streptavidin conjugates with far-red fluorophores to minimize autofluorescence interference. Apply rigorous controls including single-antibody staining to verify specific labeling patterns, absorption controls with immunizing peptides, and colocalization coefficient analysis (Pearson's or Mander's coefficients) for quantitative assessment. Super-resolution microscopy techniques such as STED or STORM can provide enhanced spatial resolution when examining membrane-bound transporters that may be in close proximity. For brain tissue sections, perform Z-stack acquisition to analyze colocalization in three dimensions, as membrane transporter distribution can vary through the depth of the tissue .

What approaches are effective for studying SLC6A13 expression changes in disease models?

Investigating SLC6A13 expression changes in disease models requires integrating multiple methodological approaches. For quantitative analysis of protein expression, Western blot using SLC6A13 antibodies normalized to housekeeping proteins provides reliable relative quantification. Complement protein expression data with mRNA analysis through qRT-PCR to determine whether changes occur at transcriptional or post-transcriptional levels. For spatial analysis, immunohistochemistry with biotin-conjugated SLC6A13 antibodies combined with digital image analysis allows region-specific and cell-type-specific quantification. In neurodegenerative disease models (particularly glaucoma models, given the disease associations of SLC6A13), double-labeling with cell-type-specific markers (e.g., GFAP for astrocytes, Iba1 for microglia) can reveal cell population-specific changes. For functional correlation, combine expression analysis with electrophysiological measurements of GABA transport or microdialysis to measure extracellular GABA levels. Longitudinal studies across disease progression stages are particularly valuable, as SLC6A13 expression may dynamically change during pathological processes. Finally, intervention studies using pharmacological agents or genetic approaches (siRNA knockdown or CRISPR-mediated editing) can establish causal relationships between SLC6A13 expression changes and disease phenotypes .

How can SLC6A13 antibodies be employed in studying drug effects on GABA transport systems?

SLC6A13 antibodies provide valuable tools for investigating pharmacological modulation of GABA transport systems. In drug discovery pipelines, these antibodies can be used in high-content screening assays to evaluate how compounds affect SLC6A13 expression, localization, and trafficking. For mechanistic studies, combine immunoprecipitation using SLC6A13 antibodies with mass spectrometry to identify drug-induced changes in post-translational modifications or protein-protein interactions. In cellular models, use live-cell imaging with fluorescently-tagged secondary antibodies against biotin-conjugated SLC6A13 primary antibodies to monitor real-time trafficking responses to drug treatment. For in vivo pharmacology studies, immunohistochemistry in brain sections from drug-treated animals can reveal region-specific effects on transporter expression. To assess drug effects on transporter function, correlate antibody-based expression data with functional GABA uptake assays using radiolabeled GABA or fluorescent GABA analogs. Dose-response relationships should be established by testing multiple concentrations over different time courses. Finally, for translational relevance, validate findings from animal models using human tissue samples (where available) stained with species-cross-reactive SLC6A13 antibodies to confirm conservation of drug effects across species .

How do various commercial SLC6A13 antibodies compare in terms of species reactivity and applications?

Commercial SLC6A13 antibodies show considerable variation in species reactivity and validated applications. Based on manufacturer data from multiple sources, comparison of key commercial antibodies reveals important differences:

When selecting between these options, researchers should prioritize antibodies validated for their specific application and species of interest. Cross-species reactivity can be particularly valuable for comparative studies between model organisms and human samples. The biotin-conjugated variant from AFG Scientific offers advantages for detection sensitivity in ELISA but has more limited species reactivity than some alternatives. For novel applications or species not listed in manufacturer validation data, preliminary validation experiments are strongly recommended before proceeding with full-scale studies .