SLC6A13 Antibody, FITC conjugated

What is SLC6A13 and what is its significance in neurological and immunological research?

SLC6A13, also known as GAT-2 (GABA transporter 2), is a sodium- and chloride-dependent GABA transporter belonging to the solute carrier family 6. This membrane protein plays a crucial role in regulating peripheral GABAergic mechanisms and has significant implications in both neurological function and immunological responses .

Recent research has revealed that SLC6A13 is physiologically and pharmacologically important as a high selectivity/low affinity transporter that demonstrates resistance to inhibition by typical GABAergic inhibitors . The transporter has a molecular weight of approximately 68-82 kDa, depending on post-translational modifications .

Beyond its neurological functions, SLC6A13 has been implicated in infection responses. Studies with Slc6a13-deficient mice have demonstrated increased resistance to Pasteurella multocida infection through mechanisms involving altered amino acid metabolism, particularly glycine accumulation, which modulates macrophage-mediated inflammatory responses .

What are the primary applications of FITC-conjugated SLC6A13 antibodies in experimental research?

FITC-conjugated SLC6A13 antibodies are versatile tools primarily designed for fluorescence-based detection methods. While ELISA is the most commonly validated application across commercial antibodies , these fluorescent conjugates are particularly valuable for:

• Immunofluorescence microscopy to visualize SLC6A13 localization in tissues and cells

• Flow cytometry for quantitative analysis of SLC6A13 expression in cell populations

• High-content imaging analysis to study protein trafficking and membrane integration

The fluorescein isothiocyanate (FITC) conjugation provides green fluorescence (excitation ~495 nm, emission ~519 nm), making these antibodies compatible with standard FITC filter sets in fluorescence microscopy and flow cytometry systems .

For optimal results in immunofluorescence applications, researchers should consider using appropriate blocking reagents to minimize non-specific binding and include proper controls to account for potential autofluorescence, particularly in tissues with high endogenous fluorescence.

What are the storage and handling recommendations for maintaining SLC6A13 antibody, FITC conjugated activity?

Proper storage and handling of FITC-conjugated SLC6A13 antibodies are critical for maintaining their activity and specificity. Based on manufacturer recommendations:

Most commercial preparations are supplied in liquid form containing preservatives and stabilizers:

• 50% glycerol buffer systems to prevent freeze-thaw damage

• PBS (pH 7.4) as the base buffer

• Preservatives such as 0.03% Proclin 300 or 0.02% sodium azide

Critical handling guidelines include:

• Avoid repeated freeze-thaw cycles which can denature the antibody and reduce FITC fluorescence intensity

• Protect from prolonged light exposure as FITC is susceptible to photobleaching

• Store in small aliquots to minimize freeze-thaw cycles for frequently used antibodies

• Allow the antibody to equilibrate to room temperature before opening to prevent condensation inside the vial

How can researchers effectively validate the specificity of SLC6A13 antibodies in experimental systems?

Validating antibody specificity is crucial for obtaining reliable results, particularly for polyclonal antibodies like the SLC6A13 FITC conjugates. A comprehensive validation strategy should include:

• Positive and negative controls:

  • Tissues/cells known to express SLC6A13 (positive control)

  • SLC6A13 knockout tissues/cells or siRNA knockdown samples (negative control)

  • Competing peptide blocking experiments using the immunogenic peptide (amino acids 1-40 for many commercial antibodies)

• Multiple detection methods:

  • Compare results across techniques (western blot, immunofluorescence, ELISA)

  • Expected molecular weight confirmation (~68 kDa for human SLC6A13)

  • Subcellular localization pattern (membrane-enriched for SLC6A13)

• Cross-reactivity assessment:

  • Testing in multiple species if working with non-human models

  • Evaluating potential cross-reactivity with other GABA transporters (GAT-1, GAT-3)

  • Utilizing computational epitope analysis to predict potential cross-reactivity

• Antibody validation metrics:

  • Signal-to-noise ratio optimization through titration experiments

  • Reproducibility assessment across different lots

  • Correlation of protein levels with mRNA expression data

Commercial SLC6A13 antibodies are typically validated through affinity purification methods (>95% purity by Protein G purification) , but independent validation in the researcher's specific experimental system remains essential.

What methodological approaches can be employed to investigate SLC6A13's role in amino acid metabolism using knockout models?

Investigation of SLC6A13's role in amino acid metabolism requires a multifaceted approach, informed by recent findings with Slc6a13-deficient mice. Based on current research, the following methodological framework is recommended:

• Experimental design considerations:

  • Use age and sex-matched Slc6a13-/- (KO) and wild-type (WT) mice

  • Include both baseline (uninfected/unstimulated) and challenged conditions

  • Control for circadian variations in amino acid metabolism

• Comprehensive amino acid profiling:

  • Liquid chromatography-mass spectrometry (LC-MS) for quantitative analysis of free amino acids in tissues of interest

  • Focus particular attention on glycine levels, which have been shown to be substantially increased in Slc6a13-deficient mice

  • Analyze both intracellular and extracellular (serum, CSF) amino acid concentrations

• Transcriptomic analysis:

  • RNA-seq to identify differentially expressed genes (DEGs) between KO and WT conditions

  • KEGG pathway analysis to identify enriched amino acid metabolism pathways

  • Validation of key findings using qRT-PCR for selected genes

• Functional assays:

  • Measure expression of glycine transporters (GlyT, especially Slc6a9) and receptors (GlyR, particularly Glra4)

  • Conduct glycine supplementation experiments to determine if exogenous glycine can recapitulate the phenotype observed in Slc6a13-deficient mice

  • Analyze inflammatory markers (IL-1β, TNF-α, IFN-γ, IL-6, IL-12) to correlate amino acid changes with functional outcomes

• Metabolic flux analysis:

  • Use isotope-labeled amino acids to track metabolic pathways

  • Measure enzyme activities involved in glycine metabolism

This approach has revealed that in infection models, Slc6a13 deficiency significantly alters amino acid metabolism pathways, with glycine accumulation playing a key role in attenuating inflammatory responses .

What are the optimal protocols for using FITC-conjugated SLC6A13 antibodies in immunofluorescence and flow cytometry applications?

For optimal results when using FITC-conjugated SLC6A13 antibodies in fluorescence-based applications, researchers should follow these protocol recommendations:

Immunofluorescence Protocol:

• Sample preparation:

  • For fixed cells: 4% paraformaldehyde (10-15 minutes), followed by 0.1-0.2% Triton X-100 permeabilization (10 minutes)

  • For tissue sections: 4-10 μm sections, deparaffinization if needed, antigen retrieval (citrate buffer pH 6.0)

• Blocking and antibody incubation:

  • Block with 5-10% normal serum (from species not related to secondary antibody) and 1% BSA for 1 hour

  • Incubate with FITC-conjugated SLC6A13 antibody at 1:100-1:500 dilution (optimize for specific lot)

  • Incubation time: 1-2 hours at room temperature or overnight at 4°C in a humidified chamber protected from light

• Nuclear counterstaining and mounting:

  • Counterstain with DAPI (1 μg/mL) for 5-10 minutes

  • Mount with anti-fade mounting medium to prevent photobleaching

  • Store slides at 4°C in the dark

Flow Cytometry Protocol:

• Cell preparation:

  • Single cell suspension (1-5 × 10^6 cells/mL)

  • Fixation with 2% paraformaldehyde (if needed)

  • Permeabilization with 0.1% saponin for intracellular detection

• Staining procedure:

  • Block with 2% FBS in PBS for 15 minutes at 4°C

  • Incubate with FITC-conjugated SLC6A13 antibody (typically 1-5 μg per 10^6 cells)

  • Incubation time: 30-45 minutes at 4°C in the dark

  • Wash twice with flow buffer (PBS + 2% FBS)

• Controls and analysis considerations:

  • Include unstained, isotype-FITC, and single-color controls

  • Compensation for spectral overlap if multicolor panel

  • Analysis gates should account for autofluorescence in the FITC channel

Optimization Considerations:

• Titrate antibody concentration to determine optimal signal-to-noise ratio

• For tissues with high autofluorescence, consider Sudan Black B treatment (0.1-0.3% in 70% ethanol) for 10 minutes after antibody staining

• For multiplexing, ensure compatible fluorophores with minimal spectral overlap with FITC

While specific applications may require protocol adjustments, these general guidelines provide a starting point for using FITC-conjugated SLC6A13 antibodies in fluorescence-based experiments .

How can researchers investigate the relationship between SLC6A13 and inflammasome activation in immunological research?

Recent research has revealed an intriguing connection between SLC6A13, glycine metabolism, and inflammasome regulation. To investigate this relationship, researchers should consider the following comprehensive approach:

• Experimental models:

  • Utilize Slc6a13-/- mice and wild-type controls

  • Employ macrophage cell lines or primary macrophages (bone marrow-derived or alveolar)

  • Consider infection models (e.g., P. multocida) or direct inflammasome activators (e.g., LPS+ATP, nigericin)

• Inflammasome component analysis:

  • Assess expression and activation of multiple inflammasome types:

    • NALP1, NLRP3, NLRC4, and AIM2 inflammasomes

    • Caspase-1 activation (pro-form vs. cleaved form)

    • ASC speck formation using imaging techniques

• Cytokine measurements:

  • Quantify inflammasome-dependent cytokines:

    • IL-1β and IL-18 (ELISA, Western blot for pro- and mature forms)

    • TNF-α, IFN-γ, IL-6, and IL-12 as inflammasome-independent controls

  • Analyze both mRNA expression (qRT-PCR) and protein secretion (ELISA)

• Glycine manipulation experiments:

  • Supplement wild-type cells/mice with exogenous glycine (suggested dose: 2 mg/kg for in vivo studies)

  • Use glycine receptor antagonists (e.g., strychnine) to block glycine effects

  • Measure glycine levels in experimental conditions using mass spectrometry

• Mechanistic investigations:

  • Examine glycine receptor expression (particularly Glra4) in macrophages

  • Investigate ion flux (Cl-, Ca2+) in response to glycine

  • Assess mitochondrial function and ROS production

  • Evaluate potassium efflux, a common trigger for NLRP3 inflammasome activation

• Data analysis framework:

  • Compare inflammasome activation parameters between:

    • WT vs. Slc6a13-/- macrophages

    • Untreated vs. glycine-treated conditions

    • Infected vs. uninfected states

  • Correlate glycine levels with inflammasome activation markers

This systematic approach can help elucidate how SLC6A13 deficiency leads to glycine accumulation, which in turn appears to block the activation of multiple inflammasomes, thereby reducing inflammatory responses during infection .

How can researchers differentiate between SLC6A13 and other GABA transporters when using antibody-based detection methods?

Differentiating between SLC6A13 (GAT-2) and other GABA transporters (GAT-1/SLC6A1, GAT-3/SLC6A11, BGT-1/SLC6A12) presents a significant challenge in neuroscience research. A strategic approach includes:

• Antibody selection considerations:

  • Choose antibodies targeting unique epitopes with minimal sequence homology to other GABA transporters

  • Review immunogen information: antibodies generated against amino acids 1-40 of human SLC6A13 are common

  • Consider using antibodies against the C-terminal region, which tends to have greater sequence divergence among SLC6 family members

• Validation in expression systems:

  • Test antibody specificity using cells overexpressing individual GABA transporters

  • Employ siRNA knockdown or CRISPR knockout of specific transporters

  • Compare staining patterns with multiple antibodies targeting different epitopes of the same protein

• Exploitation of differential expression patterns:

  • GAT-1: predominantly in neurons

  • GAT-2: expressed in astrocytes, epithelial cells, and macrophages

  • GAT-3: mainly in glial cells

  • BGT-1: expressed in liver, kidney, and brain

• Functional discrimination approaches:

  • Combine antibody staining with functional studies using selective inhibitors:

    • NO711: selective for GAT-1

    • SNAP-5114: preferentially blocks GAT-2/3

    • NNC05-2090: selective for BGT-1

  • Correlate protein expression with functional transport activity

• Comparative analysis workflow:

  • Use multiple detection methods (IF, IHC, WB) to confirm specificity

  • Run appropriate controls with blocking peptides specific to each transporter

  • Compare results with published expression patterns from RNA-seq data

  • Consider dual labeling with cell-type specific markers

Research using structure-based approaches has revealed that GAT-2 (SLC6A13) has distinctive binding site properties that make it resistant to inhibition by typical GABAergic inhibitors, unlike other GABA transporters . This functional distinction can be exploited in experimental design to differentiate between transporters.

What are the most effective strategies for overcoming background fluorescence when using FITC-conjugated antibodies?

FITC-conjugated antibodies can face significant background challenges, especially in certain tissues. Advanced researchers should employ these strategies to optimize signal-to-noise ratios:

• Tissue-specific autofluorescence reduction:

  • Lipofuscin quenching: Treat tissues with Sudan Black B (0.1-0.3% in 70% ethanol) for 10 minutes after antibody incubation

  • Photobleaching: Pre-expose samples to intense illumination in the FITC channel

  • Chemical treatment: Use sodium borohydride (0.1% in PBS) for 10 minutes before blocking step

• Fixation and permeabilization optimization:

  • Minimize fixation time (4% PFA for 10-15 minutes is often sufficient)

  • Consider non-aldehyde fixatives for sensitive applications

  • Test different permeabilization reagents (Triton X-100, saponin, methanol)

• Blocking enhancements:

  • Include 0.1-0.3% Triton X-100 in blocking buffer to reduce non-specific membrane binding

  • Add 10% serum from the host species of the primary antibody

  • Consider specialized blocking agents for endogenous biotin or Fc receptors

  • Use Image-iT FX signal enhancer for particularly challenging samples

• Antibody dilution and incubation optimization:

  • Perform titration experiments (typically 1:100-1:500 for commercial FITC-SLC6A13 antibodies)

  • Extended washing steps (4-6 washes, 5-10 minutes each)

  • Reduce incubation temperature (4°C instead of room temperature)

• Advanced imaging adjustments:

  • Spectral unmixing for separating autofluorescence from specific signal

  • Shorter exposure times with signal averaging

  • Confocal microscopy with narrow bandpass filters

  • Consider alternative fluorophores (Alexa Fluor 488 tends to provide better signal-to-noise than FITC)

• Software-based correction:

  • Background subtraction algorithms

  • Deconvolution techniques

  • Automated thresholding methods

When working with SLC6A13 antibodies specifically, researchers should be aware that macrophages and liver tissues often present significant autofluorescence challenges in the FITC channel, requiring particular attention to these background reduction strategies.

What are the appropriate controls and validation steps for experimental designs incorporating SLC6A13 antibody, FITC conjugated?

A rigorous experimental design using FITC-conjugated SLC6A13 antibodies requires comprehensive controls and validation steps:

• Essential antibody controls:

  • Isotype control: FITC-conjugated rabbit IgG at the same concentration as the primary antibody

  • Absorption control: Pre-incubate antibody with immunizing peptide (1-40AA of SLC6A13)

  • Secondary-only control: Omit primary antibody when using indirect detection systems

  • Knockout/knockdown validation: Test antibody in SLC6A13-/- tissues or siRNA-treated cells

• Fluorescence-specific controls:

  • Unstained samples: To establish baseline autofluorescence

  • Single-color controls: For compensation in multicolor experiments

  • Fluorescence minus one (FMO): Include all fluorophores except FITC

  • Photobleaching assessment: Measure signal degradation over repeated exposures

• Concentration and specificity validation:

  Validation Step	Method	Expected Outcome
  Antibody titration	Serial dilutions (1:100-1:2000)	Optimal signal-to-noise ratio
  Western blot correlation	Compare with IF/FACS results	Consistent detection at ~68 kDa
  Cross-reactivity assessment	Test multiple species	Reactivity matching manufacturer claims
  Subcellular localization	Colocalization with membrane markers	Membrane enrichment for SLC6A13

• Experimental validation metrics:

  • Signal intensity quantification across multiple experiments

  • Coefficient of variation calculation for replicates

  • Statistical comparison with published expression patterns

  • Correlation with mRNA expression data

• Advanced validation for specific applications:

  • For flow cytometry: Fluorescence intensity comparison with quantitative beads

  • For microscopy: Z-stack acquisition to confirm membrane localization

  • For ELISA: Standard curve with recombinant protein

  • For multiplexing: Spectral overlap assessment and compensation

Commercial SLC6A13 antibodies typically undergo purification (>95% using Protein G) , but batch-to-batch variation necessitates independent validation for each new lot, particularly for quantitative applications.

How can researchers troubleshoot common issues when working with FITC-conjugated SLC6A13 antibodies?

Researchers working with FITC-conjugated SLC6A13 antibodies may encounter several challenges. This systematic troubleshooting guide addresses common issues:

• Weak or absent signal:

  Possible Cause	Troubleshooting Approach
  Insufficient antibody concentration	Increase concentration (typically use 1:100-1:200 dilution for FITC conjugates)
  Epitope masking during fixation	Try different fixation methods (PFA vs. methanol) or antigen retrieval
  Low target expression	Validate SLC6A13 expression using qPCR or western blot
  Photobleaching	Minimize exposure to light; use antifade mounting media
  Storage degradation	Use fresh aliquots; avoid freeze-thaw cycles

• High background or non-specific staining:

  Possible Cause	Troubleshooting Approach
  Insufficient blocking	Extend blocking time; use 5-10% serum plus 1% BSA
  Cross-reactivity	Pre-absorb with immunizing peptide; use more specific antibody
  Autofluorescence	Apply Sudan Black B treatment; use tissue autofluorescence quencher
  Over-fixation	Reduce fixation time; optimize permeabilization
  Antibody concentration too high	Perform titration experiments to determine optimal concentration

• Inconsistent results between experiments:

  Possible Cause	Troubleshooting Approach
  Antibody lot variation	Standardize using positive controls; request same lot for critical experiments
  Protocol variations	Create detailed SOPs; control incubation times and temperatures
  Sample heterogeneity	Increase sample size; standardize tissue processing
  Microscope settings variation	Use calibration beads; standardize exposure settings
  Storage inconsistencies	Maintain consistent storage conditions (-20°C to -80°C)

• Unexpected subcellular localization:

  Possible Cause	Troubleshooting Approach
  Over-permeabilization	Titrate detergent concentration; reduce permeabilization time
  Fixation artifacts	Compare multiple fixation methods
  Cross-reactivity with other proteins	Validate with alternative antibodies targeting different epitopes
  Trafficking under experimental conditions	Include time-course analysis; compare with known conditions
  Non-specific binding	Increase washing steps; optimize blocking

• Solution preparation and storage issues:

  Issue	Solution
  FITC degradation	Store in small aliquots protected from light at -20°C to -80°C
  Buffer incompatibility	Ensure antibody diluent matches manufacturer's recommendation (typically PBS with 1% BSA)
  Antibody precipitation	Centrifuge before use; avoid detergents that may precipitate antibody
  Contamination	Include 0.02% sodium azide in storage buffer; use sterile technique
  Freeze-thaw cycles	Create single-use aliquots; store remaining stock at -80°C