GABRA5 Antibody

What is GABRA5 and what cellular function does it serve?

GABRA5 is the alpha-5 subunit of the heteropentameric ligand-gated chloride channels known as gamma-aminobutyric acid type A (GABA-A) receptors. These receptors are activated by GABA, the major inhibitory neurotransmitter in the brain. When activated, GABRA5-containing receptors allow the flow of chloride anions across the cell membrane following their electrochemical gradient, resulting in hyperpolarization of the neuronal membrane and inhibition of neuronal firing .

GABRA5-containing GABA-A receptors are primarily found in extrasynaptic locations and contribute significantly to tonic GABAergic inhibition, particularly in the hippocampus. These receptors play important roles in learning and memory processes .

What types of GABRA5 antibodies are commercially available for research?

Current research tools include both monoclonal and polyclonal antibodies targeting GABRA5:

These antibodies have been validated for various experimental applications and species reactivity, providing researchers with options based on their specific experimental needs.

How do I determine the optimal antibody dilution for my GABRA5 experiments?

Determining optimal antibody dilution is essential for generating reliable data. For GABRA5 antibodies, recommended dilutions vary by application:

• For Western Blot (WB): Initial testing at 1:500-1:2000 is recommended . Some validated protocols suggest 1:1000 dilution for mouse brain tissue lysate .

• For Immunocytochemistry/Immunofluorescence (ICC/IF): Starting dilutions of 1:100 with incubation for 1 hour at room temperature have been validated .

• For Immunohistochemistry (IHC): Dilutions between 1:50-1:500 are recommended .

Methodological approach:

• Perform a dilution series experiment (e.g., 1:100, 1:500, 1:1000, 1:2000)

• Include appropriate positive controls (e.g., mouse brain tissue for GABRA5)

• Include a negative control (omitting primary antibody)

• Evaluate signal-to-noise ratio at each dilution

• Select the dilution that provides the strongest specific signal with minimal background

It is essential to validate each new lot of antibody in your specific experimental system to ensure reproducibility.

What sample preparation methods yield optimal results for GABRA5 antibody experiments?

Effective sample preparation is critical for successful GABRA5 detection. For different applications, consider the following validated protocols:

For Western Blot:

• Fresh tissue samples should be immediately snap-frozen in liquid nitrogen

• Homogenize tissue in ice-cold RIPA buffer containing protease inhibitors

• For brain tissue lysates, 20 μg of total protein is typically sufficient for detection

• Include denaturing conditions (SDS and heat) to ensure proper epitope exposure

For Immunocytochemistry:

• 4% formaldehyde fixation has been validated for GABRA5 detection in neuronal cell lines

• Primary antibody incubation for 1-16 hours (1 hour at room temperature or overnight at 4°C)

• Secondary antibody incubation for 60 minutes at room temperature

For Immunohistochemistry:

• Antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may also be used

• Fresh-frozen or properly fixed tissues yield best results

• FFPE (formalin-fixed paraffin-embedded) samples require appropriate antigen retrieval methods

How can I validate the specificity of GABRA5 antibodies in my experimental system?

Antibody validation is essential to ensure experimental rigor. For GABRA5 antibodies, consider these validation strategies:

• Knockout/Knockdown Validation: Some GABRA5 antibodies have been validated using knockout models. For example, MA5-27700 has been validated as not cross-reacting with GABA A Receptor Alpha2 based on knockout validation data .

• siRNA/shRNA Knockdown: Utilize GABRA5-targeted shRNAs to create knockdown cell lines for antibody validation. Established protocols include:

  • Using lentiviral shRNA constructs

  • Selection with 1 μg/ml puromycin

  • Verification of knockdown by qPCR after 72 hours

• Block with Immunizing Peptide: Pre-incubate antibody with the immunizing peptide prior to application to confirm binding specificity.

• Multiple Antibody Approach: Use antibodies from different vendors or those recognizing different epitopes of GABRA5 to confirm staining patterns.

• Expected Molecular Weight: Confirm that the detected band corresponds to the expected molecular weight of GABRA5 (approximately 52-55 kDa) .

What controls should be included in experiments using GABRA5 antibodies?

Rigorous experimental design requires appropriate controls:

How can GABRA5 antibodies be used to investigate receptor trafficking and localization?

GABRA5-containing GABA-A receptors have distinct localization patterns, particularly in their extrasynaptic distribution. To investigate trafficking and localization:

• Subcellular Fractionation with Western Blot:

  • Separate membrane, cytosolic, and nuclear fractions

  • Detect GABRA5 in each fraction to monitor distribution

  • Compare results under different experimental conditions to assess trafficking

• High-Resolution Immunofluorescence:

  • Use confocal microscopy with GABRA5 antibodies (1:100 dilution)

  • Co-stain with synaptic markers (e.g., PSD95, gephyrin) to distinguish synaptic vs. extrasynaptic localization

  • Consider super-resolution techniques (STED, STORM) for nanoscale localization

• Live Cell Imaging:

  • Use fluorescently-tagged secondary antibodies against GABRA5 primary antibodies

  • Combine with FRAP (Fluorescence Recovery After Photobleaching) to assess mobility

  • Monitor internalization using antibodies against extracellular epitopes

• Electron Microscopy:

  • Use immunogold labeling with GABRA5 antibodies for ultrastructural localization

  • Quantify receptor density at synaptic vs. extrasynaptic sites

What approaches can resolve conflicting results when using different GABRA5 antibodies?

Researchers may encounter discrepancies when using different GABRA5 antibodies. To resolve such conflicts:

• Epitope Mapping:

  • Determine which region of GABRA5 each antibody targets

  • Different antibodies may recognize different epitopes that could be differentially exposed in various experimental conditions

  • For example, some antibodies target recombinant fragments within the 350-450 amino acid region

• Post-translational Modifications:

  • Consider whether PTMs affect epitope recognition

  • Use phospho-specific antibodies if phosphorylation is relevant

  • Evaluate glycosylation status of GABRA5 in your samples

• Subunit Assembly State:

  • Some antibodies may preferentially recognize assembled receptors versus individual subunits

  • Use native vs. denaturing conditions to assess this possibility

• Validation in Multiple Systems:

  • Test antibodies in overexpression systems

  • Validate with knockdown/knockout models

  • Confirm with orthogonal methods (e.g., mass spectrometry)

• Cross-Reactivity Analysis:

  • Assess potential cross-reactivity with other GABA-A receptor subunits

  • Some antibodies have been specifically validated for lack of cross-reactivity with other GABA-A receptor subunits

How can GABRA5 antibodies be used to investigate pathological conditions?

GABRA5 has been implicated in various pathological conditions, particularly in neurodevelopmental disorders and certain cancers. Research approaches include:

• Cancer Research:

  • GABRA5 is upregulated in aggressive MYC-driven "Group 3" medulloblastomas

  • Use GABRA5 antibodies for immunohistochemical classification of tumors

  • Investigate GABRA5 expression in patient-derived xenografts using validated antibodies

  • Monitor GABRA5 levels in response to therapeutic interventions

• Neurodevelopmental and Psychiatric Disorders:

  • Quantify GABRA5 expression in postmortem brain tissue from patients

  • Investigate regional and cellular distribution changes in disease states

  • Combine with electrophysiology to correlate expression with functional changes

• Drug Discovery and Target Validation:

  • Use GABRA5 antibodies to monitor receptor modulation by candidate compounds

  • Combine with functional assays to correlate expression with activity

  • Screen for compounds that alter GABRA5 trafficking or expression

How should I address high background or non-specific staining when using GABRA5 antibodies?

High background is a common challenge when working with GABRA5 antibodies. Consider these methodological solutions:

• Optimization Strategies for Western Blot:

  • Increase blocking concentration (5% BSA or milk)

  • Extend blocking time (2 hours at room temperature or overnight at 4°C)

  • Use more stringent washing conditions (higher salt concentration, longer washes)

  • Reduce primary antibody concentration (try 1:2000 instead of 1:1000)

  • Consider adding 0.1% Tween-20 to antibody dilution buffer

• For Immunohistochemistry/Immunofluorescence:

  • Pre-adsorb antibody with tissue powder from a species different from your sample

  • Include 0.1-0.3% Triton X-100 in blocking buffer for better penetration

  • Test different fixation methods (paraformaldehyde vs. methanol)

  • Optimize antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Consider autofluorescence quenching reagents for IF applications

• Verified Protocol Elements:

  • For ICC: Use 4% formaldehyde fixation with 1:100 antibody dilution

  • For WB: Incubate primary antibody for 16 hours at 4°C

  • For secondary antibodies: 1:100-1:200 dilution for 60 minutes at room temperature

What factors might influence GABRA5 detection levels in different experimental samples?

GABRA5 expression and detection can vary based on several factors:

• Developmental Regulation:

  • GABRA5 expression changes during development

  • Age-matched controls are essential for developmental studies

• Brain Region Specificity:

  • GABRA5 is enriched in hippocampus and specific cortical regions

  • Consider regional variation when selecting positive controls

• Cell Type Heterogeneity:

  • Primary expression in pyramidal neurons of the hippocampus

  • Mixed cell populations may dilute signal if analyzing whole tissue extracts

• Pathological Conditions:

  • Upregulated in certain cancers (e.g., Group 3 medulloblastomas)

  • Expression may change in response to neuronal activity or pathology

• Technical Considerations:

  • Sample storage conditions affect protein integrity

  • Freeze-thaw cycles can degrade epitopes

  • Fixation methods can mask epitopes differently

How can I quantitatively analyze GABRA5 expression data from antibody-based experiments?

Quantitative analysis requires rigorous approaches:

• Western Blot Quantification:

  • Use housekeeping proteins (β-actin, GAPDH) as loading controls

  • Employ densitometry software (ImageJ, LI-COR Image Studio)

  • Present data as ratio of GABRA5 to loading control

  • Use standard curves with recombinant protein for absolute quantification

• Immunohistochemistry Quantification:

  • Count positive cells as percentage of total cells

  • Measure staining intensity using calibrated imaging software

  • Consider automated image analysis for unbiased assessment

  • Use appropriate statistical methods for comparing groups

• Real-time qPCR Correlation:

  • Correlate protein levels with mRNA expression

  • Normalize GABRA5 mRNA to housekeeping genes like B2M

  • Present relative quantification values with appropriate statistics

• Single-Cell Analysis:

  • Consider flow cytometry for quantifying GABRA5 in dissociated cells

  • Use image cytometry for intact tissue sections

  • Correlate with other markers to identify cell type-specific expression patterns

How can GABRA5 antibodies contribute to understanding the role of extrasynaptic GABA-A receptors in cognition?

GABRA5-containing GABA-A receptors are primarily extrasynaptic and contribute to tonic inhibition, with implications for learning and memory:

• Methodological Approaches:

  • Combine GABRA5 immunostaining with electrophysiology to correlate expression with tonic currents

  • Use GABRA5 antibodies to track receptor redistribution during learning tasks

  • Apply super-resolution microscopy to map nanoscale changes in receptor distribution

• Experimental Paradigms:

  • Investigate changes in GABRA5 expression in memory-related brain regions after learning tasks

  • Use GABRA5 antibodies in combination with activity-dependent markers (c-Fos, Arc)

  • Correlate receptor expression with behavioral outcomes in cognitive tests

• Translational Implications:

  • Examine GABRA5 distribution in models of cognitive impairment

  • Correlate receptor modifications with cognitive enhancement or decline

  • Investigate receptor modulation in response to cognitive-enhancing drugs

What emerging techniques might enhance the utility of GABRA5 antibodies in research?

Several cutting-edge approaches could expand GABRA5 antibody applications:

• Proximity Labeling Techniques:

  • Combine GABRA5 antibodies with BioID or APEX2 systems

  • Identify proteins that interact with GABRA5 in specific cellular compartments

  • Map the GABRA5 interactome under different physiological conditions

• CRISPR-Cas9 Epitope Tagging:

  • Generate endogenously tagged GABRA5 for better antibody detection

  • Create knock-in reporters to correlate expression with function

  • Develop cell lines with tagged GABRA5 for high-throughput screening

• Single-Cell Proteomics:

  • Apply GABRA5 antibodies in mass cytometry (CyTOF)

  • Use antibody-based single-cell Western blotting

  • Integrate with single-cell transcriptomics for multi-omic analysis

• In vivo Imaging Applications:

  • Develop near-infrared fluorophore-conjugated GABRA5 antibodies for deeper tissue imaging

  • Apply antibody fragments for better tissue penetration

  • Explore PET imaging with radiolabeled antibodies for translational studies