SLC6A13 Antibody

What is SLC6A13 and why is it important for research?

SLC6A13, also known as GAT2 or GAT3 (GABA transporter 2/3), is a high-affinity gamma-aminobutyric acid (GABA) transporter that terminates GABA-mediated synaptic activity through reuptake mechanisms. This protein catalyzes the sodium and chloride-dependent transport of GABA, utilizing these ion concentration gradients for its function . Beyond GABA transport, SLC6A13 may also facilitate beta-alanine transport across cell membranes .

The significance of SLC6A13 in research extends beyond neuroscience. Recent studies have implicated SLC6A13 in immune responses, particularly during bacterial infections. For instance, SLC6A13-deficient mice show resistance to Pasteurella multocida infection, suggesting its role in modulating inflammatory responses . Additionally, SLC6A13 has been associated with pathological conditions including Traumatic Glaucoma and Neovascular Glaucoma , indicating its potential relevance for ophthalmological research.

What are the recommended applications for SLC6A13 antibodies?

SLC6A13 antibodies are valuable tools for investigating protein expression and localization across multiple experimental platforms. Based on validated applications, Western blotting (WB) represents the primary recommended application, with optimal dilutions typically ranging from 1/1000 to 1/3000, though researchers should determine optimal concentrations for their specific experimental conditions .

For effective application:

• Western blotting: Use for detecting SLC6A13 protein expression levels in tissue or cell lysates

• Immunohistochemistry: Though not specifically listed in the provided data, polyclonal antibodies can often be optimized for tissue section analysis

• Immunocytochemistry: Useful for cellular localization studies

• Co-immunoprecipitation: Can be employed for protein interaction studies

While implementing these techniques, researchers should include appropriate positive and negative controls to validate specificity, particularly when investigating novel tissue types or experimental contexts.

How should SLC6A13 antibodies be validated before experimental use?

Proper antibody validation is crucial for ensuring experimental reliability. For SLC6A13 antibodies, a multi-step validation approach is recommended:

• Western blot analysis: Verify the antibody detects a band of appropriate molecular weight (~68-72 kDa for SLC6A13). Test in tissues known to express SLC6A13 (brain, liver, kidney) alongside negative controls.

• Knockout/knockdown validation: Compare antibody reactivity between wild-type samples and those from SLC6A13-deficient models (such as the Slc6a13−/− mice mentioned in the research) . This represents the gold standard for antibody validation.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to verify that signal disappearance confirms specificity.

• Cross-reactivity assessment: Test reactivity across intended species (human, mouse, rat for the antibody in the search results) . This is particularly important when comparing results across model organisms.

• Reproducibility testing: Verify consistent results across different lots of the same antibody and alternative antibodies targeting different epitopes of SLC6A13.

What tissue distribution and subcellular localization patterns are expected for SLC6A13?

When investigating SLC6A13 expression patterns, researchers should be aware of its characteristic distribution profile:

Tissue distribution: SLC6A13 is prominently expressed in:

• Brain tissue (particularly in specific neuronal populations)

• Liver

• Kidney

• Lung tissue (as evidenced by the infection studies in the search results)

• Macrophages (suggested by the research on P. multocida infection)

Subcellular localization: As a membrane transporter, SLC6A13 primarily localizes to:

• Plasma membrane

• Possibly in vesicular compartments during trafficking

• Potentially at synaptic terminals in neuronal cells

When designing immunohistochemistry or immunocytochemistry experiments, these localization patterns serve as important controls for antibody specificity. Membrane-specific counterstains can help confirm proper localization patterns and distinguish true signal from background.

How can SLC6A13 antibodies be used to investigate inflammasome activation in infection models?

Recent research has revealed an unexpected connection between SLC6A13, glycine levels, and inflammasome activation during bacterial infections. When studying these interactions, consider the following methodological approach:

• Dual immunostaining protocol:

  • Fix tissue sections or cultured macrophages with 4% paraformaldehyde

  • Perform antigen retrieval using citrate buffer (pH 6.0)

  • Block with 5% normal serum and 0.3% Triton X-100

  • Co-incubate with anti-SLC6A13 antibody (1:1000) and antibodies against inflammasome components (NALP1, NLRP3, NLRC4, AIM2, or Caspase-1)

  • Visualize using spectrally distinct fluorescent secondary antibodies

  • Analyze colocalization using confocal microscopy

• Inflammasome activation assessment:

  • Isolate macrophages from wild-type and Slc6a13−/− mice

  • Challenge with bacterial components or live P. multocida

  • Immunoblot for inflammasome components and cleaved IL-1β

  • Perform ELISA for secreted inflammatory cytokines (IL-1β, TNF-α, IFN-γ, IL-6, IL-12)

  • Compare activation patterns between genotypes

• Glycine supplementation experiments:

  • Pre-treat cells with varying glycine concentrations (0.1-10 mM)

  • Challenge with infection or inflammasome activators

  • Use SLC6A13 antibodies to track potential changes in transporter expression or localization

  • Quantify inflammasome activation markers and cytokine production

This approach enables researchers to establish mechanistic links between SLC6A13 function and inflammatory responses, potentially revealing novel therapeutic targets for infection-associated inflammation.

What considerations should be made when using SLC6A13 antibodies in bacterial infection models?

When designing experiments to study SLC6A13's role in bacterial infections, researchers should address several methodological considerations:

• Temporal dynamics: Bacterial infection progression follows distinct phases. Sample collection for SLC6A13 immunodetection should span multiple timepoints (e.g., 8, 16, and 24 hours post-infection as described in the research) to capture dynamic changes in expression or localization.

• Cellular specificity: Since macrophages appear to be key mediators of SLC6A13's effect on infection outcomes, consider:

  • Isolating specific cell populations before antibody application

  • Using cell-type-specific markers (like F4/80 for macrophages) in co-immunostaining

  • Implementing flow cytometry with SLC6A13 antibodies to quantify expression in specific immune cell subsets

• Bacterial burden assessment:

  • Combine SLC6A13 immunostaining with fluorescence in situ hybridization (FISH) using bacteria-specific probes

  • Correlate SLC6A13 expression with bacterial loads in the same tissue sections

  • Quantify using digital image analysis to establish statistically meaningful relationships

• Controls for infection-induced changes:

  • Include appropriate time-matched uninfected controls

  • Consider heat-killed bacteria controls to distinguish between active infection and general bacterial component recognition

  • Include isotype control antibodies to confirm staining specificity in inflamed tissues, which may have increased background

This multi-faceted approach helps ensure that observed changes in SLC6A13 expression or localization are reliably connected to infection processes rather than technical artifacts.

How can researchers differentiate between SLC6A13 and other GABA transporters using antibody-based techniques?

Differentiating between SLC6A13 (GAT2/3) and other GABA transporters (GAT1, GAT4/BGT1) represents a common challenge in neurotransmitter transporter research. To achieve specific detection:

• Epitope selection verification:

  • Confirm that the SLC6A13 antibody targets regions with minimal homology to other GABA transporters

  • Review the manufacturer's specification regarding the synthesized peptide immunogen

  • Consider peptide alignment analysis to identify potential cross-reactivity

• Parallel detection method:

  • Implement simultaneous detection of multiple GABA transporters using carefully selected antibodies

  • Apply antibodies raised in different host species to enable multi-color immunofluorescence

  • Analyze co-expression or differential expression patterns

• Knockout/knockdown validation:

  • Test antibody specificity in Slc6a13−/− tissues (as used in the P. multocida studies)

  • Include positive controls of tissues known to express predominantly one GABA transporter subtype

  • Consider siRNA knockdown in cell culture systems for validation

• Functional correlation:

  • Combine immunodetection with functional transport assays

  • Correlate protein expression detected by antibodies with transport activities characteristic of different transporters

  • Use transporter-specific inhibitors to differentiate functional contributions

What experimental design is recommended for investigating SLC6A13's role in glycine metabolism?

The research highlights an unexpected connection between SLC6A13 deficiency and increased glycine levels, which contributes to attenuated inflammatory responses during P. multocida infection . To investigate this relationship:

• Glycine level quantification protocol:

  • Harvest tissues of interest (lungs, based on the infection model)

  • Process samples for metabolite extraction (e.g., methanol/chloroform extraction)

  • Quantify glycine levels using LC-MS/MS or other analytical techniques

  • Compare glycine concentrations between wild-type and Slc6a13−/− samples under both basal and infected conditions

• Glycine transporter and receptor analysis:

  • Use SLC6A13 antibodies alongside antibodies for glycine transporters (particularly SLC6A9)

  • Quantify expression levels by Western blot analysis

  • Perform real-time PCR to correlate protein abundance with transcript levels

  • Investigate potential compensatory upregulation mechanisms

• Glycine supplementation experiments:

  • Administer glycine (2 mg/kg intranasal delivery was effective in the referenced study)

  • Track changes in:

    • Bacterial loads

    • Inflammatory cytokine production

    • Tissue damage markers

    • Survival rates

• Macrophage-specific investigations:

  • Isolate macrophages from wild-type and Slc6a13−/− mice

  • Culture with varying glycine concentrations

  • Challenge with bacterial components

  • Assess inflammasome activation through Western blotting and ELISA

  • Correlate with SLC6A13 expression detected by antibodies

This comprehensive approach enables researchers to establish mechanistic connections between SLC6A13 function, glycine metabolism, and inflammatory responses, potentially revealing novel therapeutic strategies for infection-related inflammation.

What considerations should be made when optimizing immunohistochemistry for SLC6A13 in different tissue types?

Optimizing immunohistochemistry (IHC) for SLC6A13 across different tissues requires addressing several tissue-specific challenges:

• Fixation optimization:

  • For neural tissues: 4% paraformaldehyde for 24 hours preserves morphology while maintaining epitope accessibility

  • For lung tissues: Brief fixation (4-6 hours) helps prevent over-fixation that might mask epitopes in airway tissues

  • For liver and kidney: Perfusion fixation may improve antibody penetration in these metabolically active tissues

• Antigen retrieval customization:

  • Neural tissue: Citrate buffer (pH 6.0) with moderate heat (95°C for 20 minutes)

  • Lung tissue: Consider enzyme-based retrieval (proteinase K) if heat-based methods cause tissue disintegration

  • Liver/kidney: EDTA buffer (pH 8.0) may provide better results for membrane proteins

• Background reduction strategies:

  • For highly vascular tissues like lung: Include additional blocking with avidin/biotin if using biotinylated detection systems

  • For tissues with high endogenous peroxidase: Extend H₂O₂ quenching step (3% H₂O₂ for 15-20 minutes)

  • For inflammatory tissues: Include Fc receptor blocking step with appropriate normal serum

• Detection system optimization:

  • Low expression tissues: Consider tyramide signal amplification systems

  • High background tissues: Use polymer-based detection systems that require fewer steps

  • Multi-labeling experiments: Select enzyme substrates with appropriate spectral characteristics

• Validation controls:

  • Positive control tissues known to express SLC6A13 (brain, liver)

  • Negative control using Slc6a13−/− tissue sections

  • Absorption controls with immunizing peptide

  • Secondary-only controls to assess non-specific binding

What approaches can resolve weak or inconsistent SLC6A13 antibody signals?

When faced with weak or inconsistent SLC6A13 detection, consider implementing the following methodological interventions:

• Antibody concentration optimization:

  • Perform a dilution series beyond the manufacturer's recommended range (e.g., 1:500 to 1:5000)

  • Test multiple incubation times (2 hours at room temperature vs. overnight at 4°C)

  • Consider signal-to-noise ratio rather than absolute signal intensity

• Protein extraction enhancement:

  • For membrane proteins like SLC6A13, supplement standard RIPA buffer with:

    • Higher detergent concentrations (0.5-1% SDS)

    • Mild sonication to disrupt membrane microdomains

    • Longer extraction times at 4°C with gentle agitation

• Signal amplification strategies:

  • For Western blotting: Use high-sensitivity ECL substrates with extended exposure times

  • For IHC/ICC: Implement tyramide signal amplification or polymer-based detection systems

  • For challenging samples: Consider biotin-streptavidin amplification systems

• Sample preparation refinement:

  • Minimize freeze-thaw cycles of antibodies and samples

  • Process tissues rapidly after collection to prevent protein degradation

  • For fixed tissues, optimize fixation duration to prevent epitope masking

• Antibody validation and alternatives:

  • Test antibody performance with recombinant SLC6A13 protein as a positive control

  • Consider alternative antibodies that target different epitopes of SLC6A13

  • Verify reactivity across species if working with non-human models

These systematic troubleshooting approaches can help distinguish between technical limitations and biological realities of SLC6A13 expression patterns.

How should researchers quantify and analyze SLC6A13 expression data in comparative studies?

Proper quantification and statistical analysis are critical when comparing SLC6A13 expression across experimental conditions:

• Western blot quantification protocol:

  • Capture images within the linear dynamic range of detection

  • Normalize SLC6A13 signal to appropriate loading controls:

    • β-actin for general normalization

    • Na⁺/K⁺-ATPase for membrane protein fraction normalization

    • Consider multiple loading controls for validation

  • Perform densitometry using software that can distinguish specific signals from background

• Immunohistochemistry quantification approaches:

  • For DAB staining: Use color deconvolution and thresholding

  • For fluorescence: Measure mean fluorescence intensity in defined regions

  • Consider cell counting for percentage of positive cells rather than just intensity

  • Implement automated unbiased quantification software when possible

• Statistical analysis recommendations:

  • For comparing two groups: Student's t-test with appropriate normality testing

  • For multiple groups: ANOVA with post-hoc tests (Tukey's or Bonferroni)

  • For non-parametric data: Mann-Whitney U or Kruskal-Wallis tests

  • Report exact p-values rather than thresholds (e.g., p=0.023 vs. p<0.05)

• Correlation analysis with functional parameters:

  • Correlate SLC6A13 levels with:

    • GABA transport activity measurements

    • Glycine levels in tissues

    • Inflammasome activation markers

    • Clinical or phenotypic outcomes (e.g., survival rates in infection models)

• Visualization best practices:

  • Present both representative images and quantitative data

  • Include all data points in graphs (not just means and error bars)

  • Consider heat maps for complex datasets showing expression across multiple tissues or conditions

By implementing these methodological approaches, researchers can generate robust, reproducible data on SLC6A13 expression that facilitates meaningful biological interpretation.