SLC1A2 Recombinant Monoclonal Antibody
Description
Definition and Biological Significance
SLC1A2 recombinant monoclonal antibody refers to an antibody produced through recombinant DNA technology to target the SLC1A2 protein (solute carrier family 1 member 2), also known as excitatory amino acid transporter 2 (EAAT2) or GLT-1. This protein clears ~90% of synaptic glutamate, preventing excitotoxicity and maintaining neuronal health . Dysregulation of SLC1A2 is implicated in amyotrophic lateral sclerosis (ALS), epilepsy, schizophrenia, and drug addiction .
Production Methodology
Recombinant monoclonal antibodies are synthesized using advanced in vitro processes:
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Gene Isolation: SLC1A2 antibody genes are isolated from immunized animal B cells (e.g., rabbits) .
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Cloning: Genes are amplified and cloned into phage vectors for mammalian cell expression .
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Expression and Purification: Antibodies are mass-produced in cell lines (e.g., HEK293) and purified via affinity chromatography .
Validation and Performance
These antibodies undergo rigorous validation:
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Western Blot (WB): Detects SLC1A2 at 62.1 kDa in human, mouse, and rat brain lysates .
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Immunohistochemistry (IHC): Validated in paraffin-embedded brain tissues with DAB or fluorescence labeling .
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Flow Cytometry (FC): Used to quantify SLC1A2 expression in live cells .
Table 2: Example Antibody Performance Metrics
Research Applications
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Neurological Disorders:
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Drug Development: Ceftriaxone (antibiotic) enhances SLC1A2 expression, reducing opioid tolerance .
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Epigenetics: Promoter methylation in SLC1A2 may serve as a biomarker for bipolar disorder .
Technical Considerations
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Buffers: Often supplied in PBS with sodium azide (0.09%) and sucrose .
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Cross-Reactivity: Confirmed in zebrafish, cow, and dog models .
Future Directions
Current studies focus on:
Product Specs
The SLC1A2 recombinant monoclonal antibody is synthesized in vitro using a systematic process. Initially, SLC1A2 antibody genes are isolated from B cells derived from immunoreactive rabbits. These genes are amplified and cloned into phage vectors, which are subsequently introduced into mammalian cell lines. This facilitates the generation of functional antibodies in significant quantities. The resulting SLC1A2 recombinant monoclonal antibody is purified from the culture supernatant of the transfected cell lines through affinity chromatography. This antibody is capable of recognizing human SLC1A2 protein in three applications, including ELISA, WB, and FC.
SLC1A2, also known as EAAT2/GLT-1, plays a critical role in clearing excess glutamate from the synaptic cleft. This function prevents excitotoxicity and maintains proper neurotransmission and brain function. Its role in glutamate transport is fundamental to neuronal health, synaptic plasticity, and the prevention of neurological disorders associated with glutamate dysregulation.
Target Background
SLC1A2 is a sodium-dependent, high-affinity amino acid transporter. It mediates the uptake of L-glutamate and also L-aspartate and D-aspartate. It functions as a symporter, transporting one amino acid molecule together with two or three Na(+) ions and one proton, in parallel with the counter-transport of one K(+) ion. SLC1A2 also mediates Cl(-) flux that is not coupled to amino acid transport; this mechanism prevents the accumulation of negative charges due to aspartate and Na(+) symport. SLC1A2 is essential for the rapid removal of released glutamate from the synaptic cleft and for terminating the postsynaptic action of glutamate.
- Activated astrocytes with increased GLT-1 expression may exert beneficial roles in preserving cognitive function, even in the presence of amyloid-beta and neurofibrillary tangles in Alzheimer disease. PMID: 29374250
- Research has shown that rs3794087 of SLC1A2 does not lead to an increased risk of Parkinson's Disease in the Chinese Han population. PMID: 29275184
- Studies suggest that the TM4 domain of GLT-1, and potentially other glutamate transporters, undergoes a complex conformational shift during substrate translocation. This shift involves an increase in the proximity of the TM2 and TM4 domains in the inward-facing conformation. PMID: 27698371
- Research has demonstrated that the upregulation of GLT1 corrected Purkinje cell firing and motor incoordination in myotonic dystrophy. PMID: 28658620
- This study demonstrated that EAAT2 expression is enhanced in the ET dentate nucleus, in contrast to differentially reduced EAAT2 levels in the ET cerebellar cortex. PMID: 27624392
- This review summarizes the history leading up to the recognition of GLT-1a as a presynaptic glutamate transporter. PMID: 27129805
- This study provides further evidence for SLC1A2 mutations in epileptic encephalopathies and suggests a gain-of-function mechanism for this rather severe presentation. PMID: 28777935
- GLT1 was demonstrated by luciferase assay to be a target of miR-31-5p and miR-200c-3p. Both its mRNA and protein (immunohistochemistry) significantly decreased with age in liver biopsies and in hepatic centrilobular zone, respectively. PMID: 27995756
- Mutations in SLC1A2 and CACNA1A are Important Causes of Epileptic Encephalopathies. PMID: 27476654
- The results of this study suggested SLC1A2 rs3794087 may decrease the risk for Parkinson's disease in a Chinese cohort. However, these results do not support a role in the susceptibility to amyotrophic lateral sclerosis or multiple system atrophy. PMID: 27206883
- Research suggests that Abeta1-42 oligomers could cause disturbances in insulin/Akt/EAAT signaling in astrocytes. PMID: 26358886
- This study showed a lack of association between of SLC1A2 rs3794087 with the risk for essential tremor. PMID: 27456607
- Results suggest that genetic variation (rs4354668 and its haplotypes) in SLC1A2 may be involved in impaired executive function, which adds to the current body of knowledge regarding the risk of schizophrenia and the impairment of cognitive performance. PMID: 26459047
- SPAK and OSR1 are powerful negative regulators of the excitatory glutamate transporters EAAT1 and EAAT2. PMID: 26233565
- The results of the meta-analysis suggest that rs3794087 is not associated with the risk for essential tremor. Systematic review and meta-analysis. PMID: 26313486
- PPARgamma agonist pioglitazone has a role in modulating EAAT2 expression in glioma cells. PMID: 26046374
- Two recurrent SLC1A2 missense variants and one recurrent 5'-untranslated region variant were found to be associated with susceptibility to the development of bipolar disorder and schizophrenia. PMID: 25406999
- The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2. PMID: 26483543
- Splice variant EAAT2b levels are increased in populations of anterior cingulate cortex pyramidal cells in schizophrenic patients. PMID: 26057049
- Transcriptional factor yin yang 1 plays a critical role in the repressive effects of various neurotoxins, such as manganese (Mn), on EAAT2 expression. This review focuses on transcriptional epigenetics and translational regulation of EAAT2. PMID: 25064045
- Results showed that EAAT2 levels were significantly decreased in the essential tremor cerebellar cortex, in contrast to similar levels of EAAT1 levels between essential tremor cases and controls. PMID: 25391854
- The delivered miR-124 increased the expression of the glutamate transporters, EAAT1 in NPCs and EAAT2 in both NPCs and astrocytes. PMID: 25036385
- Results describe EAAT1 and EAAT2 labeling in the prefrontal cortex in human postmortem control brains at the light and electron microscopic level. PMID: 25064059
- EAAT-2 glutamate transporter has a role in human liver cholestasis. PMID: 24587631
- Results statistically show that a reduction in GAD1 and SCL1A2 expression in the dorsolateral prefrontal cortex in subjects with major depressive disorder is related to a possible attenuated RAF/MEK/ERK pathway. PMID: 24652383
- Sumoylated EAAT2 localizes to intracellular compartments, whereas non-sumoylated EAAT2 resides on the plasma membrane. PMID: 24753081
- This study demonstrated an association between glutamate transporter polymorphism and early stress in influencing hippocampal gray matter volume in these patients. PMID: 24518437
- The rs3794087 genotype and allelic variants were not related with the risk for migraine in Caucasian Spanish people. PMID: 24412224
- SNP rs3794087 is not related to risk for restless legs syndrome. PMID: 24424098
- IL-1beta treatment of AEG-1-overexpressing astrocytes significantly lowered expression of excitatory amino acid transporter 2. PMID: 24855648
- SLC1A2 does not play a role in susceptibility to essential tremor. PMID: 24139280
- Findings suggest important roles for up-regulated EAAT2 in chronic brain ischemia, especially in the regulation of high-affinity of extracellular glutamate and minimization of white matter damage. PMID: 23602887
- The results showed that single nucleotide polymorphism rs3794087 was associated with essential tremor among the Taiwanese. PMID: 23951268
- This study suggests that SLC1A2 rs3794087 is not associated with the risk for developing familial essential tremor in the Spanish population. PMID: 23949322
- This study demonstrated an association between polymorphisms in the glutamate transporter SLC1A2 and Parkinson's disease. PMID: 23390085
- The results of this study showed that the expression of The membrane transporters SLC1A2 and SLC1A3 was diminished in the major depressive disorder group compared to controls. PMID: 23706640
- SLC1A2 transports neurotoxic glutamic acid, which causes major mental illnesses. (review) PMID: 24334928
- Major depressive disorder is associated with unhealthy astrocytes in the noradrenergic Locus Coeruleus, characterized by a reduction in astrocyte glutamate transporter expression. PMID: 23415275
- Decreased EAAT2 protein expression alters glutamate buffering and reuptake in superior temporal gyrus and hippocampus in patients with schizophrenia. PMID: 23356950
- We provided confirmation of an association of SLC1A2 intronic variant (rs3794087) with Essential tremor in the Chinese population. PMID: 23596072
- The expression of EAAT2 in pyramidal neurons during human brain development may contribute to cortical vulnerability to excitotoxicity during the critical period for perinatal hypoxic-ischemic encephalopathy. PMID: 22522966
- Increased EAAT2 expression can protect against status epilepticus-induced death, neuropathological changes, and chronic seizure development. PMID: 22513140
- In lithium-untreated biopolar patients, we found a significant effect of genotype on the total episode recurrence rate. PMID: 23023733
- The presence of the G allele is associated, among patients with schizophrenia, with a disadvantageous effect on core cognitive functions that depend on prefrontal cortex activity. PMID: 22728822
- Research shows an association between EAAT2 protein expression in the human nucleus accumbens and a genetic polymorphism of EAAT2. PMID: 22750157
- Decreased SLC1A2 is associated with sporadic amyotrophic lateral sclerosis. PMID: 22903028
- SLC1A2, encoding the major glial high-affinity glutamate reuptake transporter in the brain, has been identified as a potential essential tremor susceptibility gene. PMID: 22764253
- GLT-1 endocytosis is independent of its phosphorylation and that Nedd4-2 mediates PKC-dependent down-regulation of the transporter. PMID: 22505712
- RNA editing in pre-mRNA EAAT2 appears to activate a cryptic alternative polyadenylation site, generating retention transcripts at a novel site in intron 7 of EAAT2. PMID: 21569822
- Evidence against cellular internalization in vivo of NMO-IgG, aquaporin-4, and excitatory amino acid transporter 2 in neuromyelitis optica. PMID: 22069320
Q&A
What is SLC1A2 and why is it an important target for recombinant antibody development?
SLC1A2 (Solute Carrier Family 1 Member 2), also known as EAAT2 (Excitatory Amino Acid Transporter 2) or GLT1 (Glutamate Transporter 1), is a sodium-dependent glutamate/aspartate transporter that plays a critical role in glutamatergic neurotransmission. It functions primarily to clear glutamate from the synaptic cleft, preventing excitotoxicity .
SLC1A2 represents an important research target because:
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It is the predominant glutamate transporter in the CNS
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Dysfunction is implicated in various neurological disorders
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Its expression is primarily astrocytic, making it valuable for studying neuron-glia interactions
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Recombinant antibodies against SLC1A2 enable precise localization and quantification studies
Recombinant monoclonal antibodies targeting SLC1A2 provide significant advantages over traditional hybridoma-derived antibodies, including better reproducibility, reduced batch-to-batch variation, and the ability to engineer specific properties for specialized applications .
What applications are SLC1A2 recombinant antibodies validated for?
SLC1A2 recombinant antibodies have been validated for multiple research applications:
When using these antibodies for specific applications, it's essential to optimize conditions for your particular experimental system, as factors such as sample preparation method and tissue type can influence antibody performance .
How should researchers properly store and handle SLC1A2 recombinant antibodies?
Proper storage and handling are critical for maintaining antibody performance:
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Storage Conditions: Store lyophilized antibodies at -20°C for up to one year . Once reconstituted, aliquot and store at -20°C to avoid repeated freeze-thaw cycles.
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Reconstitution Protocol:
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Briefly centrifuge the vial before opening
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Reconstitute in sterile water or appropriate buffer to desired concentration
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Allow complete dissolution by gentle mixing; avoid vigorous shaking
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Prepare small aliquots to prevent freeze-thaw damage
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Working Solution Stability: For immunofluorescence applications, diluted antibody solutions are typically stable for 1-2 weeks at 4°C when supplemented with 0.02% sodium azide.
Researchers should note that repeated freeze-thaw cycles can significantly reduce antibody performance. For critical experiments, validation of antibody activity after storage is recommended .
What controls should be included when using SLC1A2 recombinant antibodies?
Proper experimental controls are essential for interpreting results obtained with SLC1A2 recombinant antibodies:
Positive Controls:
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Brain tissue or astrocyte cultures with known SLC1A2 expression
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Recombinant SLC1A2 protein (for ELISA or Western blot)
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Cells transfected with SLC1A2 expression constructs
Negative Controls:
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Tissues from SLC1A2 knockout models
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Cells treated with validated SLC1A2 siRNA (e.g., siRNA sequence: 5′-CCCUGGGUCGUGUCAGGAA-3′)
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Secondary antibody-only controls
Specificity Controls:
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Pre-absorption with immunizing peptide
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Comparison with other validated SLC1A2 antibodies
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Multiple application validation (consistent results across WB, IF, IHC)
These controls help ensure that observed signals represent true SLC1A2 detection rather than non-specific binding or technical artifacts, which is particularly important in tissues with complex cellular architecture .
What strategies exist for generating antibody fragments of SLC1A2 recombinant antibodies?
Antibody fragments offer advantages in specific applications due to their smaller size and unique properties. Several formats can be generated from SLC1A2 recombinant antibodies:
Types of Antibody Fragments:
| Fragment Type | Size | Structure | Advantages | Applications |
|---|---|---|---|---|
| scFvC | ~60 kDa | VH-linker-VL + partial constant region | Increased stability over scFv | IF, WB |
| scFv | ~25 kDa | VH-linker-VL | Small size, tissue penetration | Super-resolution microscopy, live-cell imaging |
| Fab | ~50 kDa | VH-CH1 + VL-CL | Monovalent binding, reduced steric hindrance | Super-resolution microscopy |
Generation Methodology:
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For scFvC and scFv: PCR amplify variable regions, add flexible linker sequence, and clone into expression vector
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For Fab: Express truncated heavy and light chains in mammalian cells
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For direct fluorescent labeling: Add site-specific tags for conjugation or express as fusion with fluorescent proteins
These smaller fragments are particularly valuable for super-resolution microscopy of SLC1A2, where the ~10-15 nm size of full antibodies can limit achievable resolution . The use of directly labeled antibody fragments has been shown to improve spatial resolution by reducing the distance between the fluorophore and the target epitope .
How can researchers convert SLC1A2 antibody fragments back into full-length antibodies?
Converting SLC1A2 antibody fragments (such as scFv) into full-length, bivalent antibodies can be valuable for applications requiring signal amplification. The process involves:
Conversion Protocol:
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Extract heavy chain variable (VH) and light chain variable (VL) regions from the antibody fragment
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Clone VH onto species-specific heavy chain constant regions (CH1-CH2-CH3)
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Clone VL onto species-specific light chain constant regions (CL)
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Add appropriate signal peptides to both constructs
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Co-transfect both plasmids into mammalian expression cells (e.g., Expi293F)
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Harvest and purify secreted antibodies using Protein A Sepharose
This approach has been demonstrated successfully for converting single-chain antibody fragments into full-length antibodies while maintaining epitope specificity . The resulting bivalent antibodies provide advantages in standard applications:
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Enhanced signal in indirect immunofluorescence through secondary antibody amplification
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Improved sensitivity in Western blotting
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Better performance in immunoprecipitation experiments
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Increased avidity through bivalent binding
Researchers should validate that the conversion process preserves the original epitope specificity through appropriate control experiments .
What considerations are important when using SLC1A2 recombinant antibodies in super-resolution microscopy?
Super-resolution microscopy with SLC1A2 antibodies requires specific considerations to achieve optimal resolution:
Probe Selection:
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Smaller probes provide better spatial resolution
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antibody fragments (scFv, Fab) are preferable to full IgG antibodies
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Direct labeling eliminates displacement from secondary antibodies
Sample Preparation:
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Use minimal fixation (e.g., 4% PFA for 10-15 minutes)
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Employ gentle permeabilization (0.1-0.2% Triton X-100)
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Include thorough blocking to minimize non-specific binding
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For multi-color imaging, consider sequential labeling approaches
Fluorophore Considerations:
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For STORM/PALM: Use bright photoswitchable dyes (e.g., Alexa Fluor 647)
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For STED: Select dyes with good depletion properties (e.g., ATTO647N)
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Maintain low labeling ratio (1-2 fluorophores per antibody) to preserve functionality
The use of directly labeled antibody fragments has been shown to improve spatial resolution by reducing the "linkage error" between the fluorophore and the target epitope, enabling more precise localization of SLC1A2 in complex neural tissues .
How do different fixation methods affect SLC1A2 epitope recognition by recombinant antibodies?
Fixation methodology significantly impacts the detection of SLC1A2 by recombinant antibodies:
| Fixation Method | Effectiveness for SLC1A2 | Advantages | Limitations |
|---|---|---|---|
| 4% Paraformaldehyde | Good | Preserves most epitopes and membrane structure | May require permeabilization for antibody access |
| Methanol | Variable | Provides permeabilization without detergents | May disrupt conformational epitopes |
| Glutaraldehyde | Poor-Moderate | Superior ultrastructural preservation | Often masks epitopes requiring retrieval |
Optimization Recommendations:
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Test multiple fixation protocols in parallel
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For immunofluorescence, 4% PFA for 10-15 minutes at room temperature typically preserves SLC1A2 epitopes
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Post-fixation permeabilization with 0.1-0.2% Triton X-100 usually enables antibody access
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For difficult samples, mild antigen retrieval may help expose masked epitopes
Different recombinant antibodies targeting different SLC1A2 epitopes may have different fixation requirements, so optimization is recommended when first working with a new antibody .
What are the technical considerations for using SLC1A2 antibodies in multiplexed immunofluorescence experiments?
Multiplexed detection involving SLC1A2 requires careful planning to avoid cross-reactivity and ensure proper signal detection:
Species Compatibility Strategies:
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Use antibodies from different host species when possible
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Leverage species-converted recombinant SLC1A2 antibodies for flexibility
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Consider directly conjugated antibodies to eliminate secondary antibody cross-reactivity
Protocol Optimization:
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Determine optimal primary antibody concentration through titration
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Establish proper blocking conditions to minimize background
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For sequential staining, establish optimal order of antibody application
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Include appropriate controls for each antibody individually
Advanced Multiplexing Approaches:
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Tyramide signal amplification (TSA) for using multiple antibodies from same species
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Direct labeling of SLC1A2 antibodies with spectrally distinct fluorophores
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Use of smaller antibody fragments for improved spatial resolution in crowded environments
The ability to convert SLC1A2 antibodies to different species specificities (mouse, rabbit, human) provides significant flexibility in experimental design, allowing co-staining with other antibodies regardless of their species origin .
