SLC1A2 Antibody, Biotin conjugated

What is SLC1A2 and why is it an important target for neuroscience research?

SLC1A2 (also known as EAAT2 or GLT-1) is a trimeric transporter essential for clearing glutamate from neuronal synapses. As the principal transporter removing excitatory neurotransmitter glutamate from the extracellular space, it prevents neuronal damage from excessive activation of glutamate receptors. Its dysfunction has been implicated in epilepsy, amyotrophic lateral sclerosis, and other neurological disorders .

SLC1A2 research is particularly valuable because:

• It mediates the bulk of glutamate clearance in the brain via astrocytic expression

• Pan-knockout or astrocyte-specific knockout of Slc1a2 in mice results in neuronal excitotoxicity, epilepsy, and premature death

• Recurrent de novo SLC1A2 missense variants cause a severe, early-onset developmental and epileptic encephalopathy

Understanding SLC1A2 function has direct implications for developing treatments for epilepsy and other excitotoxicity-related neurological disorders.

What experimental applications are optimal for biotin-conjugated SLC1A2 antibodies?

Biotin-conjugated SLC1A2 antibodies are particularly useful for:

ELISA Applications:

• Quantitative measurement of SLC1A2 in serum, plasma, cell culture supernatants, tissue homogenates, and other biological fluids

• Sandwich ELISA formats where the biotin conjugation enables sensitive detection systems

Immunoprecipitation:

• Capturing SLC1A2 protein complexes for studying protein-protein interactions

• Investigating the interaction between SLC1A2 and other proteins like STIM1/Orai1

Western Blotting with Enhanced Detection:

• When used with streptavidin-HRP systems for enhanced sensitivity

• Detecting both monomeric (60-70 kDa) and multimeric (130-150 kDa) forms of SLC1A2

Recommended dilutions generally range from 1:500-1:5000 for Western blot applications, although optimal concentration should be determined empirically for each specific experimental setup .

How can researchers distinguish between monomeric and multimeric forms of SLC1A2 in experimental samples?

Distinguishing between different forms of SLC1A2 requires specific technical approaches:

Western Blot Analysis:

• Monomeric SLC1A2 typically appears at 55-70 kDa (different sources report slightly different sizes)

• Multimeric/dimeric forms appear at approximately 130-150 kDa

• Use gradient gels (5-20% SDS-PAGE) run at 70V (stacking gel) followed by 90V (resolving gel) for optimal separation

Sample Preparation:

• Membrane protein isolation is crucial for accurate detection

• Utilize sulfo-NHS-SS-biotin (1.5mg/ml) treatment for 1 hour at 4°C to isolate plasma membrane-bound proteins

• Include both reducing and non-reducing conditions to assess multimeric structures

Controls and Validation:

• Use peptide blocking experiments to confirm antibody specificity

• Include brain tissue lysates (mouse or rat) as positive controls

• Pre-incubation of SLC1A2 antibody with SLC1A2 peptide should eliminate the SLC1A2 band on Western blot, confirming specificity

Researchers should be aware that alternative splicing and differential glycosylation may affect the observed molecular weight of SLC1A2 in different experimental contexts .

What are the key considerations for using SLC1A2 antibodies in studying epilepsy-related mutations?

When studying SLC1A2 mutations implicated in epilepsy, researchers should consider:

Mutation-Specific Approaches:

• Three recurrent de novo SLC1A2 missense variants (Gly82Arg, Leu85Pro, and Pro289Arg) have been directly linked to epilepsy

• These mutations localize to the trimerization domain (TM2 and TM5) of SLC1A2 and affect protein function through dominant negative mechanisms

Experimental Design:

• Include wild-type SLC1A2 alongside mutant constructs to assess dominant negative effects

• Measure both protein expression/localization and functional glutamate transport

• Assess trimerization capacity using appropriate biochemical techniques

Functional Assays:

• Glutamate uptake assays are essential to determine transporter functionality

• Assess plasma membrane localization using biotinylation techniques

• Investigate protein-protein interactions, particularly with STIM1/Orai1 which may be disrupted by epilepsy-associated variants

Translational Relevance:

• Consider testing SLC1A2-modulating agents (e.g., ceftriaxone) in your experimental system

• Disease models should account for the developmental aspects of SLC1A2-associated epilepsy

How should researchers optimize immunohistochemistry protocols for SLC1A2 detection in brain tissue?

For optimal IHC detection of SLC1A2 in brain tissue:

Tissue Preparation:

• Use paraffin-embedded sections of brain tissue with heat-mediated antigen retrieval

• Optimal antigen retrieval should be performed in EDTA buffer (pH 8.0)

Blocking and Antibody Incubation:

• Block with 10% goat serum to reduce non-specific binding

• Incubate with anti-SLC1A2 antibody at 2-5 μg/ml concentration overnight at 4°C

• For biotin-conjugated antibodies, use streptavidin-based detection systems

Detection Systems:

• For chromogenic detection: use HRP-conjugated secondary reagents with DAB as the chromogen

• For fluorescent detection: use appropriate streptavidin-conjugated fluorophores with nuclear counterstaining (DAPI)

Controls:

• Include known positive controls (mouse or rat brain tissue)

• Peptide competition controls to verify specificity

• Negative controls (omitting primary antibody)

Recommended dilutions for IHC applications typically range from 1:20-1:200, though this should be optimized for each specific tissue and fixation method .

What are the methodological approaches for investigating SLC1A2 mutations using cell culture models?

To effectively study SLC1A2 mutations in cell models:

Cell Line Selection:

• HEK293 cells are well-established for studying transiently expressed SLC1A2

• Consider astrocyte cell lines for more physiologically relevant contexts

Transfection and Expression:

• Optimize transfection using Lipofectamine 2000 or similar reagents following manufacturer's protocols

• Co-transfect wild-type and mutant SLC1A2 to assess dominant negative effects

• Create stable cell lines for long-term studies

Functional Assays:

• Glutamate transport activity assays to measure transporter function

• Protein half-life determination using cycloheximide chase experiments

• Plasma membrane isolation using biotinylation techniques

Protein Interaction Studies:

• Co-immunoprecipitation to assess interactions with partner proteins

• Study interactions with STIM1/Orai1-mediated store-operated Ca²⁺ entry (SOCE) machinery

Data Analysis:

• Quantify expression using densitometry (e.g., with ImageJ software)

• Calculate transporter activity as a percentage of wild-type function

• Assess statistical significance using appropriate tests

How do researchers address cross-reactivity concerns when using SLC1A2 antibodies across different species?

When working with SLC1A2 antibodies across species:

Sequence Homology Assessment:

• Human EAAT2 shares approximately 96% amino acid sequence identity with both mouse and rat EAAT2

• Identify conserved epitopes when selecting antibodies for cross-species applications

Validation Strategies:

• Test antibodies in known positive control tissues from each species

• Perform Western blot analysis comparing human, mouse, and rat samples

• Use knockout/knockdown controls when available

Species-Specific Considerations:

• Different molecular weights may be observed between species

• Post-translational modifications may vary between species

• Expression patterns may differ between species and developmental stages

Technical Controls:

• Include peptide blocking controls for each species

• Validate antibody specificity using recombinant proteins from target species

• Consider using multiple antibodies targeting different epitopes

Some commercially available antibodies have been validated for multiple species (e.g., human, mouse, rat), but species reactivity should always be empirically confirmed for your specific application .

What are the critical parameters for optimizing Western blot protocols when detecting SLC1A2?

For optimal SLC1A2 detection via Western blot:

Sample Preparation:

• For brain tissue: use 30 μg of sample under reducing conditions

• For membrane proteins: employ biotinylation techniques to isolate plasma membrane-bound proteins

• Consider using specialized buffers containing 150mM NaCl, 5mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, and 50mM Tris-HCl pH7.4 with protease inhibitors

Gel Electrophoresis:

• Use 5-20% gradient SDS-PAGE gels for optimal separation

• Run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

Transfer Conditions:

• Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

• PVDF membranes may also be suitable for certain applications

Blocking and Antibody Incubation:

• Block with 5% non-fat milk/TBS for 1.5 hours at room temperature

• Incubate with primary antibody at appropriate dilution (typically 0.5 μg/mL for purified antibodies) overnight at 4°C

• Wash with TBS-0.1% Tween 3 times, 5 minutes each

Detection:

• Use goat anti-rabbit IgG-HRP secondary antibody (1:5000 dilution) for 1.5 hours at room temperature

• Develop with enhanced chemiluminescent detection kit

• For biotin-conjugated antibodies, use streptavidin-HRP systems

Expected band sizes: 60-70 kDa for monomeric SLC1A2 and 130-150 kDa for multimeric forms .

How can researchers investigate the relationship between SLC1A2 and STIM1/Orai1 in calcium signaling pathways?

To study SLC1A2's role in calcium signaling:

Co-immunoprecipitation Approaches:

• Use GLT-1 as the capture antibody to assess interaction with STIM1 and Orai1

• Alternatively, use STIM1 as the capture antibody to assess interaction with GLT-1 or Orai1

• Compare wild-type GLT-1 with epilepsy-associated variants (G82R, L85P, P289R)

Membrane Protein Analysis:

• Assess total and membrane Orai1 expression in the presence of wild-type vs. mutant SLC1A2

• Quantify changes in protein interaction strengths using densitometry

Functional Calcium Imaging:

• Measure store-operated Ca²⁺ entry (SOCE) in cells expressing wild-type vs. mutant SLC1A2

• Investigate whether SLC1A2 variants disturb STIM1/Orai1-mediated SOCE machinery in the endoplasmic reticulum

Structural Analysis:

• Examine whether epileptic variants affect the relative motion between transmembrane domains (particularly TM2 and TM5)

• Assess how these structural changes impact interactions with STIM1/Orai1

This approach addresses the emerging understanding that GLT-1 may be a new partner of SOCE, and disease-associated variants may reduce SOCE activity .

What strategies should researchers employ when designing experiments to study SLC1A2 in epilepsy models?

For effective epilepsy model experimental design:

Animal Model Selection:

• Consider SLC1A2 variant knock-in mice that recapitulate human mutations (G82R, L85P, P289R)

• Include randomization and blinding in experimental design

• Account for potential sex differences in SLC1A2 expression and function

Phenotypic Assessments:

• Monitor for hyperactivity and seizure-like behaviors

• Perform EEG recordings to detect epileptiform activity

• Assess developmental milestones in models of developmental and epileptic encephalopathy

Molecular Analyses:

• Quantify glutamate transporter expression using Western blot and immunohistochemistry

• Measure glutamate uptake in brain tissue preparations

• Perform electron microscopy to assess synaptic ultrastructure

Therapeutic Intervention Testing:

• Test SLC1A2-modulating agents (e.g., ceftriaxone) at various developmental timepoints

• Consider timing of intervention, as early initiation may be more efficacious

• Monitor both acute effects on seizures and long-term developmental outcomes

Translational Considerations:

• Design experiments that can inform human clinical trials

• Include assessments relevant to comorbidities (cognitive, behavioral)

• Consider pharmacological interventions that could overcome dominant negative effects of mutant SLC1A2

Research suggests there is a critical threshold of functional SLC1A2 protein (between 0-50% of wild-type) below which seizures develop, making dosage an important experimental consideration .