GABRG2 Antibody

What is GABRG2 and why is it significant in neuroscience research?

GABRG2 is a critical subunit of GABA A receptors, the major inhibitory neurotransmitter receptors in the brain. Located in the cell membrane and primarily expressed in the brain and retina, GABRG2 plays an essential role in inhibitory neurotransmission. When activated by GABA, these receptors allow chloride anions to flow across the cell membrane, decreasing the neuron's ability to generate action potentials and thereby reducing nerve transmission .

GABRG2 is particularly important because:

• Mutations in the GABRG2 gene are associated with various forms of epilepsy, including childhood absence epilepsy, febrile seizures, and epileptic encephalopathy

• The γ2 subunit is necessary for the formation of synaptic contacts

• GABRG2-containing receptors are found at both synaptic and extrasynaptic sites, contributing to both phasic and tonic inhibition

• It plays a crucial role in the synaptogenic activity of GABA A receptors when combined with α1 and β2 or β3 subunits

What applications have been validated for GABRG2 antibodies?

Based on the research literature, GABRG2 antibodies have been validated for multiple applications:

These applications have been documented in numerous publications, with at least 8 studies using WB, 4 using IF, 2 using IHC, and 1 using IP as noted in the search results .

What molecular weight should I expect when detecting GABRG2?

When detecting GABRG2 with antibodies, researchers should be aware of several molecular weight considerations:

• Calculated molecular weight: 54 kDa (467 amino acids)

• Observed molecular weight range: 54-60 kDa in most Western blot applications

• Additional bands that may be detected:

  • 43 kDa band (reported in PMID:12091434)

  • Multiple bands at 25 kDa, 42 kDa, and 51 kDa have been observed with certain antibodies

  • The 51 kDa band is hypothesized to represent the mature form of GABRG2

This variability in molecular weight can be attributed to post-translational modifications, particularly glycosylation. For example, the I107T mutation introduces a new glycosylation motif (NXS/T) in the extracellular domain, resulting in a higher molecular weight band compared to wild-type GABRG2 .

How should I optimize GABRG2 antibody dilutions for different applications?

Optimal antibody dilution is critical for specific detection of GABRG2. Based on the literature, recommended dilutions vary by application:

For Western Blotting:

• The general recommended range is 1:500-1:2000

• Start with a mid-range dilution (e.g., 1:1000) and adjust based on signal-to-noise ratio

• For mouse brain tissue, successful detection has been reported at various dilutions within this range

For Immunoprecipitation:

• Use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate

• The amount may need adjustment based on GABRG2 expression levels in your sample

For Immunofluorescence:

• Begin with a 1:50-1:500 dilution range for paraffin-embedded sections

• Higher concentrations (1:50-1:100) may be needed for weakly expressed regions

• Lower concentrations (1:200-1:500) may be sufficient for regions with high expression

It's emphasized that "this reagent should be titrated in each testing system to obtain optimal results" as outcomes can be "sample-dependent" . Consider performing a dilution series during initial optimization.

What controls are essential when using GABRG2 antibodies?

Proper controls are crucial for validating GABRG2 antibody specificity and ensuring reliable results:

• Negative controls:

  • Tissue from GABRG2 knockout models (e.g., Gabrg2+/- or Gabrg2 fl/wt Cre+ mice)

  • These models show significantly reduced GABRG2 immunostaining in specific brain regions

  • Secondary antibody-only controls to assess non-specific binding

• Positive controls:

  • Tissues with confirmed GABRG2 expression:

    • Mouse brain tissue, particularly cerebellum (Purkinje cell layer)

    • Neuro-2a cells

  • Recombinant GABRG2 expression systems

• Validation approaches:

  • Cross-validation with different GABRG2 antibodies targeting distinct epitopes

  • Correlation with mRNA expression data

  • Epitope blocking with immunizing peptide (e.g., GABRG2 fusion protein Ag5237)

  • Comparison between wild-type and heterozygous animals (e.g., Gabrg2+/Q390X mice)

Quantitative analysis of staining intensity across different brain regions (e.g., hippocampus CA1, CA3, DG regions and neocortex) can further validate the specificity of staining patterns .

How can I properly handle and store GABRG2 antibodies?

The stability and performance of GABRG2 antibodies depend on proper handling and storage:

Storage conditions:

• Store at -20°C in buffer containing PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Antibodies remain stable for approximately one year after shipment when properly stored

• Aliquoting may not be necessary for -20°C storage for some antibody preparations

• Some formulations (20μl sizes) contain 0.1% BSA as a stabilizer

Working solutions:

• Prepare fresh dilutions on the day of experiment when possible

• For multi-day experiments, store diluted antibody at 4°C with appropriate preservatives

• Avoid repeated freeze-thaw cycles of the stock solution

• Allow reagents to reach room temperature before opening to prevent condensation

Safety considerations:

• Note that most preparations contain sodium azide, which is toxic and should be handled accordingly

• Proper disposal procedures should be followed according to institutional guidelines

How can I use GABRG2 antibodies to study epilepsy-associated mutations?

GABRG2 antibodies are invaluable tools for investigating the molecular mechanisms of epilepsy-associated mutations:

• Expression level analysis: Western blotting can detect altered GABRG2 expression in mutant models. For example, studies have shown that both heterozygous knockout (Gabrg2+/-) and point mutation (Gabrg2+/Q390X) mouse models show distinct patterns of GABRG2 expression .

• Subcellular localization: Immunofluorescence reveals changes in GABRG2 distribution. The Q390X mutation causes increased γ2 subunit staining in somatic regions across different ages, suggesting consistent ER retention of the mutant protein .

• Surface expression quantification: Surface biotinylation combined with Western blotting can measure changes in GABRG2 trafficking. Research on de novo mutations (A106T, I107T, P282S, R323Q, R323W, F343L) revealed specific reductions in surface levels:

  Mutation	Surface Level Reduction	Statistical Significance
  A106T	0.74 ± 0.03	P < 0.05, n = 6
  I107T	0.76 ± 0.06	P < 0.05, n = 6
  P282S	0.65 ± 0.02	P < 0.05, n = 4
  R323Q	0.73 ± 0.07	P < 0.05, n = 5
  R323W	0.46 ± 0.09	P < 0.05, n = 6
  F343L	0.53 ± 0.05	P < 0.05, n = 6

  These reductions (24-54%) correlated with reductions in whole-cell currents, linking protein expression to functional deficits .

• Protein complex analysis: Some mutations (P282S, I107T) cause formation of high molecular mass protein complexes (∼75–150 kD), which may represent oligomers of mutant GABRG2 .

• Mutation mapping: Structural modeling helps understand how mutations affect protein function. For instance, the six de novo mutations identified in epileptic encephalopathy patients were mapped to locations closely connected among structural domains between the N-terminal and transmembrane domains .

How can I study GABRG2 trafficking and localization in neurons?

Multiple approaches can be used to study GABRG2 trafficking and localization:

• Subcellular fractionation: This technique separates cellular components to determine GABRG2 distribution within different compartments. The search results mention protocols for isolating synaptosomes to study GABRG2 localization at synapses .

• Immunofluorescence microscopy: This approach enables visualization of GABRG2 distribution patterns in brain tissue:

  • In wild-type mice, GABRG2 shows specific distribution patterns in the cerebellum, particularly in the Purkinje cell layer

  • In Gabrg2+/Q390X mice, increased γ2 subunit staining is observed in somatic regions, indicating ER retention

  • Quantitative analysis can reveal region-specific changes in GABRG2 expression, as demonstrated in the hippocampus (CA1, CA3, DG regions) and neocortex of Gabrg2 fl/wt Cre+ mice

• Surface biotinylation: This method specifically labels and isolates proteins at the cell surface. In studies of GABRG2 mutations, surface biotinylation revealed that all six de novo mutations reduced surface expression, with the most severe effects seen with R323W (54% reduction) and F343L (47% reduction) .

• Tagged constructs: Epitope-tagged GABRG2 constructs (e.g., HA-tagged or FLAG-tagged) can facilitate trafficking studies by enabling specific detection of exogenous protein .

What approaches can I use to study GABRG2 in functional assays?

Combining antibody-based detection with functional assays provides deeper insights into GABRG2 biology:

• Electrophysiology correlation: GABRG2 expression levels detected by antibodies can be correlated with electrophysiological measurements. For example, research has shown that reductions in surface GABRG2 expression correlate with decreased GABA-evoked currents .

• Zinc sensitivity assays: The presence of the γ2 subunit in GABA A receptors confers decreased sensitivity to zinc inhibition. Antibody detection of GABRG2 can be paired with zinc sensitivity assays to assess functional incorporation into receptors .

• Behavioral phenotyping: Correlating GABRG2 expression patterns with behavioral outcomes provides functional context. For instance, the Gabrg2 fl/wt Cre+ mice with neocortex and hippocampus-specific deletion reproduce many features of febrile seizures, providing a model for studying region-specific GABRG2 functions .

• Pharmacological manipulation: Combining antibody detection with pharmacological interventions can reveal functional roles of GABRG2 in specific neural circuits.

• Co-expression systems: For in vitro studies, co-expressing GABRG2 with α1 and β2 subunits in HEK293T cells creates functional receptors that can be assessed both biochemically and electrophysiologically .

Why might I observe multiple bands or unexpected molecular weights with GABRG2 antibodies?

Multiple factors can explain the detection of multiple bands or unexpected molecular weights:

• Post-translational modifications: GABRG2 undergoes glycosylation, which can affect its migration pattern. The search results mention that GABRG2 has "glycosylation modification" .

• Mutation effects: Some mutations can introduce new glycosylation sites. For example, the I107T mutation creates a new glycosylation motif (NXS/T) resulting in a shift in molecular mass .

• Protein complex formation: Certain mutations (P282S, I107T) can cause formation of high molecular mass protein complexes (∼75–150 kD) that appear as additional bands on Western blots .

• Processing variants: The 43 kDa band mentioned in one study may represent a processed form of GABRG2 .

• Isoform detection: Different GABRG2 isoforms (e.g., γ2S and γ2L mentioned in the search results) may have slightly different molecular weights .

• Degradation products: Improper sample handling or storage may result in degradation fragments being detected.

When analyzing Western blot results, researchers should consider these possibilities and include appropriate controls to distinguish between specific and non-specific bands.

How should I quantify GABRG2 expression changes in experimental models?

Accurate quantification of GABRG2 expression requires careful methodological approaches:

What special considerations apply when working with GABRG2 mutants?

The search results highlight several important considerations when studying GABRG2 mutations:

• Heterozygous vs. homozygous models: Many GABRG2 mutations exhibit haploinsufficiency, with phenotypes present in heterozygous states. Studies have used Gabrg2+/- knockout and Gabrg2+/Q390X models to investigate heterozygous effects .

• Region-specific effects: GABRG2 mutations may affect different brain regions differently. The Gabrg2 fl/wt Cre+ model shows specific deletion in neocortex and hippocampus while sparing other regions like the olfactory bulb and brain stem .

• Developmental timing: Consider analyzing samples across different developmental stages, as some mutations show age-dependent effects. The γ2 subunit staining pattern in Gabrg2+/Q390X mice has been examined at both P0 and 16 months of age .

• Structural impacts: Mutations in different domains (N-terminal, transmembrane domains M1-M3) can affect protein function and trafficking differently. Structural modeling can help predict these effects .

• Dominant-negative effects: Some mutations like the intronic IVS6+2T→G mutation exert dominant-negative effects on receptor assembly, reducing surface αβγ2 receptor levels .

• Co-expression with other subunits: Since functional GABA A receptors require multiple subunits, GABRG2 should be studied in the context of receptor assembly. Many studies co-express GABRG2 with α1 and β2 subunits .

How can GABRG2 antibodies be used to explore novel therapeutic approaches for epilepsy?

GABRG2 antibodies offer potential for developing and evaluating new therapeutic strategies:

• Identifying trafficking enhancers: Antibodies can help screen compounds that correct trafficking defects of mutant GABRG2 proteins. By measuring surface expression changes, researchers could identify molecules that restore proper localization of mutant proteins.

• Evaluating gene therapy approaches: Antibodies are essential for assessing the efficacy of gene therapy interventions targeting GABRG2. They can measure whether therapeutic approaches successfully restore normal GABRG2 expression patterns.

• Monitoring treatment responses: In animal models of GABRG2-related epilepsy, antibodies can track changes in protein expression following treatment with antiepileptic drugs or experimental therapies.

• Precision medicine approaches: Antibodies could help characterize patient-specific GABRG2 variants in cellular models, enabling personalized therapeutic strategies.

• Biomarker development: Research could explore whether GABRG2 expression patterns correlate with clinical outcomes, potentially leading to prognostic biomarkers.

The Gabrg2 fl/wt Cre+ mouse model, which reproduces many features of febrile seizures, provides a valuable platform for testing such therapeutic approaches .

What emerging techniques might enhance GABRG2 antibody applications in research?

Several cutting-edge approaches could extend the utility of GABRG2 antibodies:

• Super-resolution microscopy: Techniques like STORM or STED could reveal nanoscale details of GABRG2 localization at synapses that conventional microscopy cannot resolve.

• Live-cell imaging: Combining antibody fragments with fluorescent proteins could enable real-time tracking of GABRG2 trafficking in living neurons.

• Proximity labeling: Techniques like BioID or APEX2 fused to GABRG2 could identify novel interaction partners in specific subcellular compartments.

• Single-cell analysis: Combining antibody-based detection with single-cell transcriptomics could reveal cell type-specific GABRG2 expression patterns.

• CRISPR-based approaches: CRISPR/Cas9-mediated tagging of endogenous GABRG2 could enable more physiological studies of the protein without overexpression artifacts.

• Patient-derived models: GABRG2 antibodies could help characterize expression in induced pluripotent stem cell (iPSC)-derived neurons from patients with epilepsy.

These approaches could significantly advance our understanding of GABRG2 biology and its role in neurological disorders.