slc17a6b Antibody

What is SLC17A6/SLC17A6B and what is its function in neural tissue?

SLC17A6 (Solute Carrier Family 17 Member 6), also known as VGLUT2 (Vesicular Glutamate Transporter 2), mediates the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells . The protein plays a crucial role in glutamatergic neurotransmission, which is essential for normal brain function and development. Additionally, research suggests that SLC17A6 may also mediate the transport of inorganic phosphate . The protein has a molecular weight of approximately 64 kDa and has been identified in various regions of the brain, particularly in glutamatergic neurons.

What are the key differences between polyclonal and monoclonal antibodies against SLC17A6?

Polyclonal SLC17A6 antibodies, such as the rabbit polyclonal antibody targeting amino acids 255-289, recognize multiple epitopes on the SLC17A6 protein . This characteristic provides higher sensitivity but potentially lower specificity. Monoclonal antibodies like the mouse monoclonal S29-29 clone bind to a single epitope (often in the AA 501-582 region), offering higher specificity but sometimes lower sensitivity . For experiments requiring precise localization or quantification, monoclonal antibodies may be preferred, while polyclonal antibodies are advantageous for initial detection or applications with low target abundance.

How can SLC17A6 antibodies be used to identify glutamatergic neurons in brain tissue?

SLC17A6 antibodies serve as reliable markers for glutamatergic neurons in immunohistochemistry and immunofluorescence experiments . When preparing brain tissue samples, optimal results are achieved using 4% paraformaldehyde fixation followed by cryoprotection in sucrose before sectioning. For immunofluorescence, researchers typically dilute primary SLC17A6 antibodies 1:200-1:500 in blocking buffer containing 0.1% Triton X-100 and incubate overnight at 4°C . Co-staining with neuronal markers like NeuN can help confirm specificity. For quantitative analysis, confocal microscopy with appropriate controls should be employed to accurately identify glutamatergic neurons.

How can I optimize double immunolabeling protocols using SLC17A6 antibodies with other neuronal markers?

When performing double immunolabeling with SLC17A6 antibodies and other neuronal markers, sequential staining protocols often yield better results than simultaneous application . Begin by validating single staining for each antibody separately. For optimization, consider these parameters:

For co-labeling with TH (tyrosine hydroxylase) or GAD1, which identify dopaminergic and GABAergic neurons respectively, careful optimization is necessary as these markers identify distinct neuronal populations that sometimes show anticorrelation patterns with SLC17A6 .

What expression correlation patterns exist between SLC17A6 and other neuronal marker genes?

Analysis of in situ hybridization data from the Allen Brain Atlas reveals significant correlation patterns between SLC17A6 and other neuronal markers . Research shows that SLC17A6 expression profiles positively correlate with 171 genes uniquely, while showing significant anticorrelations with other neuronal markers . Specifically:

• SLC17A6 shows anticorrelation with 68 GAD1-associated genes and 12 TH-associated genes

• P2ry14 has been identified as having similar expression patterns to SLC17A6

• Wwox shows expression patterns similar to SLC17A6 and overlaps with TH expression areas

These correlation patterns are particularly valuable for researchers studying neuron subtype differentiation and can help identify co-expression patterns relevant to glutamatergic neuron function . When designing experiments to investigate these relationships, using multiple marker genes rather than relying solely on SLC17A6 will provide more robust characterization of neuronal populations.

How reliable are SLC17A6 antibodies compared to in situ hybridization for identifying glutamatergic neurons?

In situ hybridization (ISH) and antibody-based immunohistochemistry (IHC) each offer distinct advantages for identifying glutamatergic neurons through SLC17A6/VGLUT2 detection . ISH directly detects mRNA expression and thus avoids potential cross-reactivity issues inherent in antibody-based detection methods. The Allen Brain Atlas ISH data for SLC17A6 provides high spatial resolution of expression patterns that have been used to identify correlations with other neuronal markers .

• Using both techniques in parallel for initial characterization

• Employing ISH for mRNA expression pattern analysis across brain regions

• Utilizing antibodies for detailed subcellular localization and protein-level studies

• Validating antibody specificity using knockout/knockdown controls when possible

The correlation between mRNA expression (ISH) and protein detection (IHC) can itself be informative about post-transcriptional regulation of SLC17A6.

What are the critical validation steps needed before using a new lot of SLC17A6 antibody?

Before implementing a new lot of SLC17A6 antibody in critical experiments, proper validation is essential to ensure experimental reproducibility. The recommended validation protocol includes:

• Western blot analysis to confirm specificity at the expected molecular weight (~64 kDa)

• Positive control tissue with known SLC17A6 expression (e.g., specific brain regions)

• Negative control tissue lacking SLC17A6 expression

• Peptide competition assay to verify epitope specificity

• Comparison with previous antibody lot in parallel experiments

Document all validation results, including images of western blots and immunostaining patterns. For polyclonal antibodies like ABIN6242124, lot-to-lot variation may be more significant than with monoclonal antibodies, making thorough validation particularly important .

How should I troubleshoot weak or non-specific signals when using SLC17A6 antibodies?

When encountering weak or non-specific signals with SLC17A6 antibodies, systematic troubleshooting is necessary. Consider the following approach:

For flow cytometry applications, the recommended antibody dilution is approximately 1:25, significantly higher than for western blotting . Always include appropriate positive and negative controls to facilitate troubleshooting interpretation.

What storage and handling practices maximize SLC17A6 antibody stability and performance?

To ensure optimal performance and longevity of SLC17A6 antibodies, proper storage and handling practices are essential:

• Store antibodies according to manufacturer specifications, typically at 4°C for short-term (up to 1 month) or -20°C for long-term storage

• Avoid repeated freeze-thaw cycles by aliquoting the antibody upon receipt

• Use sterile technique when handling antibody solutions to prevent contamination

• For working dilutions, use fresh buffer containing a stabilizing protein (BSA) and sodium azide (0.09% W/V) to prevent microbial growth

• Monitor for signs of degradation such as precipitates or decreased performance

• Document the date opened and use within the expiration period (typically 6 months from reconstitution)

Purified antibodies like ABIN6242124 are supplied in PBS with sodium azide as a preservative . While this extends shelf life, note that sodium azide is hazardous and should be handled accordingly. Additionally, sodium azide can inhibit HRP activity in certain applications, so thorough washing is required before detection steps in HRP-based assays.

How are SLC17A6 antibodies being used in studies of neuronal circuit mapping?

SLC17A6 antibodies have become valuable tools in comprehensive neuronal circuit mapping studies. By enabling the specific identification of glutamatergic neurons, these antibodies help researchers understand excitatory circuit architecture. Modern applications include:

• Array tomography with SLC17A6 antibodies for high-resolution 3D reconstruction of glutamatergic synapses

• Correlative light and electron microscopy (CLEM) to combine ultrastructural details with specific labeling

• Multiplexed immunofluorescence with other synaptic markers to identify specific connection types

• Clearing-enhanced 3D imaging (using techniques like CLARITY or iDISCO) with SLC17A6 antibodies for whole-brain glutamatergic neuron mapping

The expression correlation patterns between SLC17A6 and other genes like P2ry14 and Wwox provide additional markers that may be used in combination for more precise circuit analysis . These approaches have revealed important insights into the organization of glutamatergic projections in various brain regions.

What machine learning approaches can enhance antibody-antigen binding prediction for SLC17A6 research?

Recent advances in machine learning offer promising approaches to predict antibody-antigen binding, which could improve SLC17A6 antibody development and application. Active learning strategies can significantly enhance prediction efficiency by reducing the number of required experimental samples . Key approaches include:

• Library-on-library screening approaches that analyze many-to-many relationships between antibodies and antigens

• Out-of-distribution prediction models that can work with novel antibody-antigen pairs not represented in training data

• Iterative active learning algorithms that can reduce required antigen variants by up to 35%

• Simulation frameworks like Absolut! that allow for evaluation of prediction performance

These computational approaches can accelerate antibody development by prioritizing the most informative experiments, potentially leading to more specific and effective SLC17A6 antibodies . When implementing these methods, researchers should consider both the computational requirements and the integration with existing experimental workflows.

What criteria should guide the selection between different SLC17A6 antibody options?

When selecting an SLC17A6 antibody for specific research applications, several key criteria should be considered to ensure optimal results:

For advanced applications like multiplex immunostaining, direct conjugates (FITC, PE, APC) may be preferable to avoid cross-reactivity issues . The purification method (protein A column followed by peptide affinity purification) also affects specificity and should be considered when comparing antibody options .

How do SLC17A6 expression patterns compare across different brain regions and developmental stages?

SLC17A6 expression shows distinct patterns across brain regions and developmental stages, which are important considerations when designing experiments. Based on in situ hybridization data from the Allen Brain Atlas and antibody studies:

• Regional differences: SLC17A6 expression is particularly prominent in the ventral tegmental area (VTA), thalamus, and specific cortical layers

• Developmental regulation: Expression increases during early postnatal development in parallel with synaptogenesis

• Co-expression patterns: In the VTA, SLC17A6 shows partial overlap with TH (dopaminergic marker), indicating a population of glutamatergic-dopaminergic neurons

• Subcellular localization: Primarily localized to synaptic vesicles at presynaptic terminals

Understanding these expression patterns is critical for designing proper controls and interpreting experimental results. When studying mixed neuronal populations, the correlation and anticorrelation patterns with other markers (like the observed anticorrelations with 68 GAD1-associated genes) can help in proper identification of cell types .