GABRA4 Antibody

What is GABRA4 and what is its significance in neurological research?

GABRA4 is the gene encoding the alpha 4 subunit of the GABAA receptor, a heteropentameric ligand-gated chloride channel activated by gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain. GABAA receptors function as chloride channels that, when activated by GABA, allow chloride anions to flow across the cell membrane down their electrochemical gradient . GABRA4-containing receptors are primarily located extrasynaptically and contribute to tonic inhibition in dentate granule cells and thalamic relay neurons, controlling levels of excitability and network activity .

GABRA4's significance in neurological research stems from its involvement in the etiology of autism spectrum disorders, as indicated by multiple studies . Additionally, GABAA receptors containing alpha-4-beta-3-delta subunits can simultaneously bind GABA and histamine, potentially playing a role in sleep-wake regulation . This makes GABRA4 a valuable target for studying inhibitory neurotransmission, neuronal excitability, and neurological disorders.

What are the different types of GABRA4 antibodies available for research?

Based on the search results, several types of GABRA4 antibodies are available for research applications, each with specific characteristics:

These antibodies target different epitopes of the GABRA4 protein. For example, the extracellular antibody targets amino acid residues 37-50 of rat GABRA4 at the N-terminus , while others may target different regions. The selection of a specific antibody should be based on the intended application, the species being studied, and the particular epitope required for detection.

What are the recommended applications and dilutions for GABRA4 antibodies?

GABRA4 antibodies can be used in various applications, with specific recommended dilutions for optimal results:

It is important to note that these dilutions serve as starting points, and researchers should optimize conditions for their specific experimental systems. As stated in the product information, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" . Different tissue types or experimental conditions may require adjustment of antibody concentrations.

In which tissues and cells has GABRA4 expression been confirmed?

GABRA4 expression has been confirmed in multiple tissues and cell types across different species:

This expression pattern indicates GABRA4's widespread presence in the nervous system, with notable expression in the brain, particularly in regions involved in inhibitory neurotransmission. The expression in non-neuronal tissues like kidney and skeletal muscle suggests additional functions outside the nervous system that might be worth investigating.

What are the optimal storage conditions for GABRA4 antibodies?

For maintaining GABRA4 antibody activity and stability, the following storage conditions are recommended:

• Store at -20°C

• Antibodies are typically stable for one year after shipment when properly stored

• Aliquoting is generally unnecessary for -20°C storage

• Antibodies are usually provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some preparations (20μl sizes) may contain 0.1% BSA

Proper storage is critical for maintaining antibody function. Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of antibody activity. While aliquoting is noted as unnecessary according to the manufacturer, it may still be beneficial for antibodies that will be used repeatedly over extended periods.

How can specificity of GABRA4 antibodies be validated in knockout models?

Validation of GABRA4 antibodies in knockout models is a critical step for ensuring specificity and reliability of experimental results. Based on the search results, several approaches have been used:

The most rigorous validation involves comparing antibody reactivity in wild-type versus GABRA4 knockout mice. The search results describe various knockout models:

• Constitutive α4-subunit knockout mice (α4−/−) generated by crossing heterozygote mice carrying the loxP-flanked gabra4 gene

• Conditional knockout mice with deletion localized to specific neuronal populations using Cre-loxP technology:

  • α4D1−/− (deletion in DRD1 neurons)

  • α4D2−/− (deletion in DRD2 neurons)

• Viral knockdown using adenoviruses carrying shRNA designed to knock down α4 (Ad-shα4) compared with scrambled sequence controls (Ad-NSS)

Validation protocols should include:

• Western blot analysis comparing wild-type and knockout tissues

• Quantitative PCR to confirm reduction in Gabra4 mRNA levels

• Immunohistochemistry comparing staining patterns in wild-type and knockout tissues

• Use of appropriate negative controls including pre-absorption with the immunizing peptide

When using viral knockdown approaches, researchers should verify knockdown efficiency through qPCR analysis of the target region, as described in the research where "RNA was extracted from biopsy punches (1 × 1 mm) of the NAc and dorsal striatum... and subjected to reverse transcriptase in the presence of oligo(dT), and the resulting cDNA was used in a qPCR reaction to amplify Gabra4" .

What are the methodological considerations for immunohistochemical detection of GABRA4 in brain tissues?

Immunohistochemical detection of GABRA4 in brain tissues requires attention to several methodological details:

• Antibody selection: Choose antibodies targeting extracellular epitopes for better access in intact tissues. The extracellular antibody targeting amino acid residues 37-50 of rat GABRA4 at the N-terminus has shown success in brain tissue staining .

• Fixation methods: While specific fixation protocols aren't detailed in the search results, standard paraformaldehyde fixation is commonly used. The fixation method should preserve the epitope targeted by the antibody while maintaining tissue morphology.

• Co-localization studies: Use neuronal markers to identify specific cell populations. For example, the search results show co-staining with parvalbumin (a marker of Purkinje and interneuronal cells) to demonstrate GABRA4 expression around the soma of Purkinje cells in rat cerebellum .

• Signal visualization: The immunohistochemical staining shown in the search results used fluorescence detection (GABRA4 in green, parvalbumin in red, and DAPI as a blue nuclear counterstain) .

• Controls: Include appropriate negative controls such as tissue from knockout animals or antibody preincubated with blocking peptide, as shown in the Western blot analysis where antibody reactivity was blocked by preincubation with GABA(A) α4 Receptor blocking peptide .

• Regional expression patterns: The search results indicate that in rat cerebellum, GABRA4 staining appears in the molecular layer and around the soma of Purkinje cells , consistent with its role in inhibitory neurotransmission in these regions.

What are the key considerations for Western blot detection of GABRA4?

Western blot analysis of GABRA4 requires specific technical considerations to obtain reliable results:

• Expected molecular weight: GABRA4 has a calculated molecular weight of 62 kDa (554 amino acids), but the observed molecular weight in Western blots typically ranges from 62-70 kDa . This variation may be due to post-translational modifications like glycosylation.

• Sample preparation: The search results indicate successful detection in various sample types:

  • Brain tissue lysates from mouse and rat

  • Brain membrane preparations

  • Human cell line lysates (CCF-STGI Brain astrocytoma)

  • Skeletal muscle tissue

  • Kidney tissue

• Antibody dilutions: For Western blot applications, recommended dilutions range from 1:500 to 1:3000 . Optimization may be necessary depending on the sample type and antibody used.

• Specificity controls: To ensure specificity, include appropriate controls:

  • Compare with knockout or knockdown samples

  • Pre-incubate antibody with the immunizing peptide (blocking peptide) to confirm specific binding

• Detection methods: Standard chemiluminescence or fluorescence-based detection systems are compatible with GABRA4 antibody detection, though specific recommendations are not provided in the search results.

• Multiple band interpretation: If multiple bands appear, careful validation is required to determine which represents specific GABRA4 signal. The search results show Western blot analysis where specific bands disappear when the antibody is pre-incubated with blocking peptide .

How can genetic manipulation approaches be used to study GABRA4 function?

The search results describe several genetic manipulation approaches for studying GABRA4 function:

• Constitutive knockout: Generation of α4−/− mice by genetic deletion of the first coding exon (exon3) of the gabra4 gene using the Cre-loxP system . These mice completely lack GABRA4 expression throughout the body.

• Conditional knockout: Cell-type specific deletion using Cre-recombinase expressed under the control of specific promoters:

  • α4D1−/− mice: GABRA4 deletion in dopamine receptor D1-expressing neurons

  • α4D2−/− mice: GABRA4 deletion in dopamine receptor D2-expressing neurons

• Viral-mediated knockdown: Local reduction of GABRA4 expression using stereotaxic delivery of adenoviruses expressing:

  • Ad-shα4: shRNA designed to knock down α4

  • Ad-NSS: scrambled sequence control

The viral knockdown approach allows for region-specific manipulation, as demonstrated by the stereotaxic infusion into the nucleus accumbens (NAc) with the following coordinates: AP 1.34, L ±1.40, DV −4.20 .

For validation of genetic manipulations, the search results describe the use of quantitative PCR to assess mRNA levels:

• RNA extraction from tissue punches

• Reverse transcription with oligo(dT) primers

• qPCR amplification of Gabra4

These genetic approaches provide powerful tools for dissecting the role of GABRA4 in specific neuronal populations and brain regions, enabling the study of its contribution to behavior, neuronal excitability, and potential role in neurological disorders.

What experimental approaches can be used to study GABRA4 in the context of autism research?

Given GABRA4's implicated role in autism etiology , several experimental approaches can be employed to investigate its function in this context:

• Genetic association studies: While not directly mentioned in the search results, genetic variants in GABRA4 can be analyzed in autism cohorts to identify potential risk alleles.

• Expression analysis in autism models: Quantitative PCR, Western blot, and immunohistochemistry can be used to assess GABRA4 expression levels in animal models of autism or in postmortem brain tissue from individuals with autism.

• Electrophysiological studies: Since GABRA4-containing receptors contribute to tonic inhibition , whole-cell patch-clamp recordings can measure changes in tonic inhibitory currents in autism models.

• Behavioral studies using genetic models: The conditional and constitutive knockout models described in the search results can be subjected to behavioral tests relevant to autism, such as social interaction, repetitive behaviors, and anxiety tests.

• Pharmacological manipulation: Since GABRA4-containing receptors respond differently to drugs than other GABAA receptor subtypes, specific modulators can be used to probe their function in autism models.

• Circuit-specific manipulation: The conditional knockout approach targeting specific neuronal populations (e.g., α4D1−/− and α4D2−/−) allows for investigation of GABRA4's role in specific neural circuits implicated in autism.

• Interaction with histamine system: Given that GABAA receptors containing alpha-4-beta-3-delta subunits can simultaneously bind GABA and histamine , investigating this interaction might provide insights into sleep abnormalities often observed in autism.

Researchers should consider combining multiple approaches to comprehensively understand GABRA4's role in autism pathophysiology.

What are the key considerations for selecting the optimal GABRA4 antibody for specific research applications?

When selecting a GABRA4 antibody, researchers should consider:

• Intended application: Different antibodies perform optimally in different applications. Based on the search results, consider:

  • For Western blot and IP: Polyclonal antibody 12979-1-AP shows good reactivity

  • For ELISA: Both polyclonal (12979-1-AP) and recombinant (83030-4-RR) antibodies are suitable

  • For immunohistochemistry: Anti-GABA(A) α4 Receptor extracellular antibody shows good results in brain tissue

  • For ICC/IF: Monoclonal antibody [S398A-34] is recommended

• Species reactivity: Ensure the antibody recognizes GABRA4 in your species of interest:

  • For human samples: All listed antibodies show reactivity

  • For rodent studies: Most antibodies react with mouse and rat samples

• Epitope location: Consider whether you need an antibody targeting an extracellular epitope (better for live cell or intact tissue studies) or intracellular epitope.

• Validation evidence: Review available validation data, especially Western blot images and control experiments. Antibodies validated in knockout models provide the highest confidence in specificity.

• Antibody format: Consider whether polyclonal, monoclonal, or recombinant antibodies best suit your needs:

  • Polyclonal: Good for detecting low-abundance proteins but may have batch-to-batch variation

  • Monoclonal: High consistency but might recognize only a single epitope

  • Recombinant: Combines consistency of monoclonals with renewable supply

Each project's specific requirements should guide the selection of the most appropriate GABRA4 antibody to ensure reliable and reproducible results.

How can researchers troubleshoot common issues with GABRA4 antibody experiments?

When troubleshooting GABRA4 antibody experiments, consider these approaches:

• Weak or no signal in Western blot:

  • Verify protein loading with housekeeping controls

  • Adjust antibody concentration (try the higher end of recommended range: 1:500)

  • Increase exposure time or protein amount

  • Verify sample preparation preserves the epitope

  • Check if the target is expressed in your sample (GABRA4 shows tissue-specific expression)

• Multiple bands or non-specific signal:

  • Include a blocking peptide control to identify specific bands

  • Optimize blocking conditions to reduce background

  • Try more stringent washing

  • Consider using tissues from knockout animals as negative controls

• Poor immunohistochemistry results:

  • Optimize fixation protocols (overfixation can mask epitopes)

  • Try antigen retrieval methods

  • Adjust antibody concentration

  • Consider using the extracellular epitope antibody for better accessibility

  • Include proper controls like pre-absorption with blocking peptide

• Failed immunoprecipitation:

  • Increase antibody amount (up to 4.0 μg for 1.0-3.0 mg of total protein)

  • Verify antibody binding conditions (temperature, buffer composition)

  • Consider the strength of antibody-antigen interaction

• Inconsistent qPCR results when validating knockdown:

  • Ensure proper primer design specific to Gabra4

  • Use appropriate reference genes for normalization

  • Verify RNA quality and cDNA synthesis efficiency

  • Follow the validated protocol described for Gabra4 amplification

Proper experimental design, including appropriate controls and optimization of protocols for specific applications, is essential for successful GABRA4 antibody experiments.

What future directions are emerging in GABRA4 research?

Based on the search results and current trends in neuroscience research, several promising future directions in GABRA4 research include:

• Cell-type specific functions: Further exploration of GABRA4's role in specific neuronal populations using conditional knockout approaches, such as the α4D1−/− and α4D2−/− models , will help delineate its function in different neural circuits.

• Role in neurodevelopmental disorders: Given GABRA4's implicated role in autism , investigating its contribution to other neurodevelopmental disorders could yield valuable insights into shared pathophysiological mechanisms.

• Interaction with histamine system: The ability of GABAA receptors containing alpha-4-beta-3-delta subunits to simultaneously bind GABA and histamine opens new avenues for investigating cross-talk between inhibitory and histaminergic systems in sleep regulation and other processes.

• Tonic inhibition in disease states: Further research into how alterations in GABRA4-mediated tonic inhibition contribute to pathological states could identify new therapeutic targets.

• Extrasynaptic versus synaptic signaling: More detailed investigation of GABRA4's extrasynaptic localization and its implications for neuronal excitability control would enhance our understanding of inhibitory neurotransmission.

• Novel therapeutic strategies: Development of subunit-selective compounds targeting GABRA4-containing receptors could offer new approaches for treating conditions associated with altered inhibitory signaling, including autism spectrum disorders.

• Sex differences: The search results mention sex as a factor in experimental analyses , suggesting that exploring sex differences in GABRA4 function could provide insights into sex-biased neurological conditions.