GABRD Antibody

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

GABRD is the gene that encodes the delta subunit of the ligand-gated chloride channel for gamma-aminobutyric acid (GABA), which functions as the major inhibitory neurotransmitter in the mammalian brain . The GABRD protein has a calculated molecular weight of 51 kDa, although it is sometimes observed at 63-66 kDa in experimental conditions due to post-translational modifications . This protein is particularly important in neuroscience research because alterations in GABRD expression or function have been implicated in various neurological disorders, and it serves as a component of extrasynaptic GABA receptors that mediate tonic inhibition. Additionally, recent evidence suggests GABRD may play roles outside the nervous system, including in cancer progression .

Methodologically, when investigating GABRD in neural tissues, researchers should consider its relatively low expression levels in certain brain regions and design experiments with appropriate sensitivity and controls.

What are the optimal applications for GABRD antibodies in laboratory research?

GABRD antibodies can be utilized across multiple experimental platforms with varying dilution requirements and optimization needs:

The methodological approach should include titration of the antibody concentration for each specific experimental system to achieve optimal signal-to-noise ratios . When using GABRD antibodies for multiple applications, validation across each platform is essential, as performance can vary significantly between applications even with the same antibody.

How should researchers interpret different molecular weights observed for GABRD protein?

The molecular weight disparities observed for GABRD require careful interpretation:

• The calculated molecular weight based on amino acid sequence is 51 kDa

• Observed molecular weights include both 51 kDa and higher bands at 63-66 kDa

• These higher molecular weight bands likely represent post-translationally modified forms of the protein

Methodologically, researchers should:

• Include molecular weight markers spanning 40-70 kDa range

• Be prepared to observe multiple bands

• Verify band specificity using peptide competition or knockdown controls

• Consider tissue-specific expression patterns that may affect band appearance

When comparing GABRD expression between different experimental conditions, consistency in sample preparation is critical to ensure that differences in band patterns reflect biological variations rather than technical artifacts.

What cross-species reactivity should be expected with GABRD antibodies?

GABRD antibodies demonstrate varying cross-reactivity profiles that must be considered when designing experiments involving multiple species:

Methodological considerations for cross-species applications include:

• Performing validation experiments in each species of interest

• Checking sequence homology of the target epitope across species

• Using positive control samples from each species

• Adjusting antibody concentrations when switching between species

When antibodies are used for species not listed in the reactivity data, additional validation steps such as western blotting with positive and negative controls from the target species are essential.

What are the optimal storage and handling conditions for maintaining GABRD antibody activity?

Proper storage and handling of GABRD antibodies are critical for maintaining their functionality:

• Store at -20°C for long-term stability

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

• The standard storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

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

For experimental handling:

• Minimize freeze-thaw cycles by creating single-use aliquots if working with large volumes

• Return antibodies to -20°C promptly after use

• Keep antibodies on ice during experiment preparation

• Centrifuge briefly before opening to collect solution at the bottom of the tube

Following these methodological approaches will help ensure consistent antibody performance across experiments and maximize shelf life.

What validation strategies should researchers employ to confirm GABRD antibody specificity?

A comprehensive validation strategy is essential for ensuring GABRD antibody specificity:

• Gene Manipulation Controls: Implement GABRD knockdown using shRNA with validated sequences such as "CAGACACCATTGACATTTA" or "CTCATTTCAACGCCGACTA" . Compare antibody staining between control and knockdown samples.

• Peptide Competition Assay: Pre-incubate the antibody with its immunizing peptide (e.g., amino acids 331-358 for ABIN653247 ) before application to samples. Signal reduction confirms specificity.

• Multiple Antibody Approach: Use antibodies targeting different GABRD epitopes and compare staining patterns. Consistent results across antibodies suggest specific detection.

• Protein-mRNA Correlation: Compare antibody staining with mRNA expression using qPCR primers:

  • Forward: 5′-GCATCCGAATCACCTCCACTG-3′

  • Reverse: 5′-GATGAGTAACCGTAGCTCTCCA-3′

• Known Expression Pattern Comparison: Compare antibody staining with established GABRD expression patterns in tissues like brain, kidney, and specific cell lines (HeLa) .

This methodological framework provides multiple independent confirmation approaches, strengthening confidence in antibody specificity and experimental results.

How should researchers optimize Western blotting protocols specifically for GABRD detection?

Optimized Western blotting protocols for GABRD detection should address several critical parameters:

• Sample Preparation:

  • Include protease inhibitors to prevent degradation

  • For brain tissue samples, rapid processing is essential

  • Consider membrane enrichment for improved signal

• Electrophoresis Parameters:

  • Use 10-12% SDS-PAGE gels for optimal resolution of 51-66 kDa proteins

  • Run at lower voltage (80-100V) to improve band resolution

  • Load 20-50 μg of total protein per lane

• Transfer Conditions:

  • Use PVDF membranes for higher protein binding capacity

  • Transfer at 30V overnight at 4°C for improved transfer of membrane proteins

• Antibody Incubation:

  • Dilute primary GABRD antibody at 1:500-1:2000

  • Incubate overnight at 4°C with gentle agitation

  • Use 5% non-fat milk or BSA in TBST for blocking and antibody dilution

• Detection Strategy:

  • Use high-sensitivity ECL substrates for visualization

  • Include GAPDH (1:2,000 dilution) as loading control

  • Be prepared to observe bands at both 51 kDa and 63-66 kDa

• Troubleshooting Considerations:

  • If background is high, increase washing steps or reduce antibody concentration

  • If signal is weak, extend exposure time or increase protein loading

  • For multiple bands, verify specificity through additional controls

This methodological approach provides a comprehensive framework for optimal GABRD detection by Western blotting.

What is known about GABRD expression in pathological versus normal tissues?

Research indicates significant differences in GABRD expression between normal and pathological states:

"GABRD expression was significantly increased in CRCs [colorectal cancers] compared to that in NTs [normal tissues], but was similar between metastasis and primary tumors" . This suggests GABRD upregulation may be associated with cancer development but not necessarily with metastatic progression.

For methodological analysis of GABRD expression in pathological contexts:

• Tissue Processing Approach:

  • Use formalin-fixed paraffin-embedded sections with sodium citrate buffer (pH 6.0) for antigen retrieval

  • Block with 3% hydrogen peroxide to inactivate endogenous peroxidase

  • Consider microwave treatment to enhance antibody binding

• Quantification Methods:

  • Implement a modified H score system for semi-quantitative analysis of IHC samples

  • Use qPCR with GABRD-specific primers for mRNA quantification

  • Apply densitometry to Western blots using appropriate normalization

• Experimental Design Considerations:

  • Include paired normal and pathological samples from the same patients

  • Stratify samples by pathological grade or stage

  • Account for potential confounding factors (age, treatment status)

This methodological framework enables rigorous comparison of GABRD expression between normal and pathological states, potentially revealing new insights into disease mechanisms.

How can GABRD knockdown or overexpression systems be optimized for functional studies?

Developing effective GABRD manipulation systems requires careful consideration of multiple methodological parameters:

• Knockdown Approaches:

  • Use validated shRNA sequences targeting GABRD: "CAGACACCATTGACATTTA", "CTCATTTCAACGCCGACTA", "TGACGATGACCACGCTCAT", or "GTTACTCATCGGAGGACAT"

  • Implement lentiviral vector systems (GV248 vector has been validated)

  • Include scramble control: "TTCTCCGAACGTGTCACGT"

  • Select stable transfectants using puromycin at 0.4 μg/mL initially, then maintenance at 0.2 μg/mL

• Overexpression Systems:

  • Utilize lentiviral plasmids expressing GABRD (GV144 vector has been validated)

  • Confirm expression by both qPCR and Western blotting

  • Use empty vector controls in parallel experiments

• Validation Methods:

  • Confirm knockdown/overexpression efficiency by qPCR using primers:

    • Forward: 5′-GCATCCGAATCACCTCCACTG-3′

    • Reverse: 5′-GATGAGTAACCGTAGCTCTCCA-3′

  • Verify protein level changes by Western blotting using anti-GABRD antibody (1:1,000 dilution)

• Functional Assays:

  • Cell proliferation assays using stably transfected cell lines

  • Migration assessment using scratch wound healing assays

  • Electrophysiological recordings for functional channel studies

This comprehensive methodological approach enables robust investigation of GABRD function through genetic manipulation systems.

What are the emerging implications of GABRD in cancer research?

Recent research has revealed significant implications for GABRD in cancer biology:

"GABRD promotes progression and predicts poor prognosis in colorectal cancer" , indicating its potential role as both a biomarker and functional contributor to cancer pathophysiology.

Key methodological approaches for investigating GABRD in cancer include:

• Expression Analysis Framework:

  • Compare GABRD levels between matched normal and cancerous tissues

  • Assess correlation with clinical outcomes and pathological parameters

  • Use multiple detection methods (IHC, qPCR, Western blotting) for comprehensive analysis

• Functional Investigation Strategy:

  • Implement both knockdown and overexpression systems to assess phenotypic changes

  • Evaluate effects on proliferation, migration, and invasion capabilities

  • Investigate downstream signaling pathways affected by GABRD modulation

• Translational Research Approaches:

  • Develop tissue microarrays for high-throughput analysis of GABRD in patient cohorts

  • Correlate expression levels with treatment response and survival outcomes

  • Explore potential as a therapeutic target or stratification marker

Experimental results indicate that GABRD expression is significantly increased in colorectal cancers compared to normal tissues , but doesn't differ between metastatic and primary tumors, suggesting its involvement in early cancer development rather than metastatic progression.

How can researchers optimize immunohistochemistry protocols for GABRD detection in fixed tissues?

Optimizing immunohistochemistry for GABRD detection requires attention to multiple critical parameters:

• Tissue Processing Protocol:

  • Use formalin fixation with controlled fixation times (24-48 hours optimal)

  • Process tissues to paraffin embedding following standard procedures

  • Section at 4-6 μm thickness for optimal antibody penetration

• Antigen Retrieval Optimization:

  • Implement sodium citrate buffer (pH 6.0) heat-induced epitope retrieval

  • Apply microwave treatment for 10 minutes as validated in published protocols

  • Allow slides to cool slowly to room temperature after heating

• Blocking Strategy:

  • Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes

  • Use protein blocking solution appropriate to the detection system

  • Include separate blocking step to reduce non-specific binding

• Antibody Incubation Parameters:

  • Optimize primary antibody dilution through titration experiments

  • Incubate at 4°C overnight for maximum sensitivity

  • Use humidity chamber to prevent section drying

• Detection System Selection:

  • Employ two-step plus Poly-HRP anti-Rabbit IgG Detection System

  • Visualize using DAB chromogen with controlled development times

  • Counterstain with hematoxylin for structural context

• Quantification Approach:

  • Implement modified H score system for semi-quantitative analysis

  • Consider digital image analysis for more objective quantification

  • Include positive and negative controls in each experiment

This comprehensive methodological framework provides a robust approach for GABRD detection in fixed tissue samples.

What methods are most effective for quantifying GABRD expression in experimental samples?

Multiple complementary approaches can be employed for quantitative assessment of GABRD expression:

• Western Blotting with Densitometry:

  • Use GABRD antibody at 1:500-1:2000 dilution

  • Include GAPDH (1:2,000 dilution) as loading control

  • Analyze band intensity using image analysis software

  • Normalize GABRD signal to loading control for relative quantification

• Quantitative PCR Method:

  • Extract RNA using validated protocols

  • Perform reverse transcription with oligo(dT) primers

  • Amplify GABRD using specific primers:

    • Forward: 5′-GCATCCGAATCACCTCCACTG-3′

    • Reverse: 5′-GATGAGTAACCGTAGCTCTCCA-3′

  • Normalize to GAPDH expression for relative quantification

• Semi-quantitative IHC Analysis:

  • Implement modified H score system based on staining intensity and distribution

  • Calculate scores by multiplying intensity (0-3) by percentage of positive cells

  • Use digital pathology platforms for more objective assessment

• Flow Cytometry Approach:

  • Prepare single-cell suspensions from tissues or cell cultures

  • Optimize staining protocol using GABRD antibodies validated for FACS

  • Analyze median fluorescence intensity as measure of expression

  • Include isotype controls to determine background staining

This multimodal approach provides comprehensive quantification of GABRD at both mRNA and protein levels, enabling robust comparative analysis across experimental conditions.

How can researchers troubleshoot non-specific binding when using GABRD antibodies?

When encountering non-specific binding with GABRD antibodies, researchers should implement a systematic troubleshooting approach:

• Antibody Dilution Optimization:

  • Test serial dilutions within and beyond the recommended range (1:500-1:2000)

  • Identify the dilution providing optimal signal-to-noise ratio

  • Document antibody lot number and optimal dilution for reproducibility

• Blocking Protocol Enhancement:

  • Extend blocking time from standard 1 hour to 2-3 hours

  • Test alternative blocking agents (5% BSA, 5% normal serum, commercial blockers)

  • Consider dual blocking with different blocking agents sequentially

• Washing Procedure Modification:

  • Increase wash buffer volume and duration

  • Add additional washing steps between critical incubations

  • Increase detergent concentration in wash buffer (up to 0.1% Tween-20)

• Sample Processing Evaluation:

  • For IHC: Optimize fixation time and antigen retrieval conditions

  • For Western blotting: Ensure complete protein denaturation

  • For all applications: Minimize endogenous enzyme activity

• Validation Control Implementation:

  • Include peptide competition controls to identify specific signal

  • Use GABRD knockdown samples prepared with validated shRNA sequences

  • Test multiple GABRD antibodies targeting different epitopes

This methodological framework provides a comprehensive approach to identifying and eliminating sources of non-specific binding when working with GABRD antibodies.

What considerations are important when selecting between polyclonal and monoclonal GABRD antibodies?

The selection between polyclonal and monoclonal GABRD antibodies should be guided by specific experimental requirements:

For GABRD research specifically:

• Several validated polyclonal antibodies are available (15623-1-AP, ABIN653247)

• Polyclonals have demonstrated successful detection of GABRD in multiple applications

• Consider using both types in parallel for critical experiments requiring high confidence

Methodological approach to antibody selection:

• Define primary application requirements

• Consider target abundance in your experimental system

• Evaluate importance of batch-to-batch consistency

• Assess available validation data for candidate antibodies

• Test multiple antibodies when possible for critical research

This structured decision framework enables optimal antibody selection based on specific experimental needs.

How can researchers integrate GABRD expression analysis into broader neuroscience or cancer research programs?

Effective integration of GABRD analysis into broader research programs requires thoughtful methodological approaches:

• Multimodal Analysis Strategy:

  • Combine protein-level analysis (WB, IHC) with transcript analysis (qPCR)

  • Correlate GABRD expression with functional outcomes

  • Integrate with -omics approaches (proteomics, transcriptomics) for pathway analysis

• Cross-disciplinary Study Design:

  • For neuroscience: Correlate GABRD expression with electrophysiological properties

  • For cancer research: Assess GABRD as potential biomarker for patient stratification

  • For both fields: Investigate signaling pathways downstream of GABRD

• Translational Research Framework:

  • Develop tissue microarrays for high-throughput GABRD assessment

  • Correlate expression with clinical outcomes and treatment responses

  • Consider GABRD as therapeutic target using knockdown approaches validated in preclinical models

• Technical Integration Approach:

  • Standardize GABRD detection methods across research platforms

  • Implement central validation to ensure consistency between research groups

  • Develop data sharing protocols for GABRD-related findings

This comprehensive methodological framework enables meaningful integration of GABRD analysis into broader research contexts, potentially revealing new insights into neurological disorders and cancer biology.