DLG3 Antibody

What is DLG3 and why is it significant in neuroscience research?

DLG3 (discs large MAGUK scaffold protein 3) encodes the SAP102 protein, a member of the membrane-associated guanylate kinase protein family. This protein plays a critical role in clustering NMDA receptors at excitatory synapses and is required for learning through its role in synaptic plasticity. DLG3 is notably expressed in the hippocampus, cerebral cortex, and cerebellum, making it a significant target in neuroscience research . Mutations in the DLG3 gene have been associated with X-linked mental retardation, highlighting its importance in cognitive development .

What are the known isoforms of DLG3 and their expression patterns?

DLG3 gives rise to approximately ten different transcripts, with four being protein-coding. Two of these transcripts (ENST00000374360.8 and ENST00000194900.8) are translated to larger isoforms (90-93 kDa) that are predominantly brain-specific and contain 19 and 21 exons, respectively. The shorter transcripts (ENST00000374355.8 and ENST00000542398.1) contain 14 and 12 exons and are translated to proteins of 58 kDa and 42 kDa, which are more widely expressed throughout different human tissues . The isoforms share their C-terminal sequence, but the N-terminal sequence is unique to the larger brain-specific isoforms .

Which applications are most commonly used with DLG3 antibodies?

DLG3 antibodies are primarily utilized in Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), and ELISA applications. Western Blot is the most widely used application, typically at dilutions between 1:500-1:10000, while IHC applications generally use dilutions between 1:100-1:1000 . These applications enable researchers to detect and quantify DLG3 protein expression in various tissues and experimental models.

How should I select the appropriate DLG3 antibody for my specific research application?

When selecting a DLG3 antibody, consider:

• Target epitope: Determine whether you need to target specific isoforms. For detecting larger brain-specific isoforms, choose antibodies targeting the N-terminal region. For detecting all isoforms, select C-terminal-targeting antibodies .

• Species reactivity: Verify the antibody's reactivity with your experimental model (human, mouse, rat, etc.) .

• Application compatibility: Ensure the antibody is validated for your intended application (WB, IHC, IF, etc.) .

• Validation data: Review available validation data, including Western blot images, IHC staining patterns, and published literature .

• Antibody type: Consider whether a polyclonal or monoclonal antibody best suits your needs based on specificity requirements and experimental design .

What are the optimal sample preparation protocols for DLG3 antibody applications?

For effective DLG3 detection, sample preparation varies by application:

Western Blot:

• Use SDS-PAGE with 7.5% gels for optimal resolution of the 90-115 kDa DLG3 protein

• Apply 20-50 μg of protein lysate per lane

• For brain tissue extracts, include protease inhibitors to prevent degradation

Immunohistochemistry:

• Perform heat-mediated antigen retrieval with 10 mM citrate buffer (pH 6.0) or 10 mM PBS buffer (pH 7.2)

• Use paraffin-embedded sections at 4-5 μm thickness

• For brain tissue, perfusion fixation with 4% paraformaldehyde yields optimal results

Immunofluorescence:

• Methanol fixation has been reported effective for DLG3 immunofluorescent staining

• Optimize blocking with 3-5% normal serum from the species of secondary antibody

What controls should be included when working with DLG3 antibodies?

How can I distinguish between DLG3 isoforms in experimental settings?

Distinguishing between DLG3 isoforms requires careful experimental design:

• Antibody selection: Use N-terminal antibodies to detect only larger isoforms (90-93 kDa) and C-terminal antibodies to detect all isoforms .

• Gel electrophoresis optimization: For Western blot analysis, use gradient gels (4-15%) to effectively separate the different molecular weight isoforms (42 kDa, 58 kDa, and 90-93 kDa).

• RNA analysis: Implement RT-PCR with isoform-specific primers targeting unique exons. The larger transcripts contain exon 1, which is absent in smaller isoforms .

• Tissue specificity: Remember that larger isoforms are predominantly brain-specific, while smaller isoforms have wider tissue distribution .

• Subcellular fractionation: Different isoforms may have distinct subcellular localizations that can be exploited for separation and identification.

What approaches can resolve discrepancies in DLG3 molecular weight observations?

Researchers frequently observe DLG3 at apparent molecular weights of 110-115 kDa despite calculated weights of 90 kDa . To resolve these discrepancies:

• Post-translational modifications: Investigate phosphorylation, glycosylation, or other modifications that may alter migration patterns using phosphatase treatment or glycosylation inhibitors.

• Sample preparation: Compare reducing vs. non-reducing conditions and various detergents to determine their impact on observed molecular weight.

• Cross-reactivity assessment: Validate specificity using knockout/knockdown samples or pre-absorption with recombinant protein.

• Mass spectrometry: Use peptide mass fingerprinting to confirm protein identity when observed MW differs from predicted values.

• Isoform analysis: Consider that observed bands may represent different splice variants or processed forms of DLG3.

How might DLG3 mutations affect antibody detection in clinical research samples?

DLG3 mutations associated with X-linked cognitive disability may impact antibody detection through:

• Truncation effects: Mutations like the stop gain variant c.195del/p.(Thr66ProfsTer55) found in the MRX20 family result in truncated proteins that may not be detected by C-terminal antibodies .

• Epitope masking: Missense mutations may alter protein folding, potentially masking epitopes recognized by certain antibodies.

• Expression level changes: Mutations affecting transcription or translation efficiency may result in reduced protein levels despite intact epitopes.

• Isoform-specific impacts: Mutations in exon 1 affect only larger brain-specific isoforms while sparing shorter isoforms, requiring careful antibody selection to detect mutation effects .

• Tissue consideration: Lymphoblastoid cell lines from patients may express DLG3 mRNA but lack detectable protein, necessitating brain tissue for conclusive analysis .

What are common issues encountered with DLG3 antibodies and how can they be resolved?

How can RNA and protein expression analyses be combined to comprehensively study DLG3?

An integrated approach to DLG3 analysis includes:

• Transcript analysis: Utilize RT-PCR and RNA-seq to identify and quantify specific DLG3 transcript isoforms. Design primers to distinguish between the larger brain-specific transcripts and shorter widely-expressed transcripts .

• Protein detection: Employ both N-terminal and C-terminal antibodies in Western blot analysis to detect different isoforms. The N-terminal antibody will detect large isoforms (90-93 kDa), while C-terminal antibodies can detect both large and small isoforms (42-58 kDa) .

• Spatial expression: Combine in situ hybridization for transcript localization with immunohistochemistry for protein distribution analysis to identify potential post-transcriptional regulation.

• Temporal dynamics: Track developmental expression patterns of both RNA and protein to identify critical periods for DLG3 function.

• Disease models: Compare RNA-seq differential expression data with protein levels in disease models to identify potential translational regulation or protein stability issues .

What methodological approaches are most effective for studying DLG3 in neuronal systems?

For neuronal systems, the following methodological approaches are most effective:

• Primary neuronal cultures: Use hippocampal or cortical neurons with immunofluorescence to visualize DLG3 subcellular localization at synapses.

• Brain slice immunohistochemistry: Employ microwave antigen retrieval with 10mM PBS buffer (pH 7.2) before IHC staining to optimize DLG3 detection in brain tissues .

• Synaptosomal fractionation: Isolate synaptic compartments to enrich for DLG3 and associate it with NMDA receptors and other binding partners.

• Proximity ligation assays: Detect in situ protein-protein interactions between DLG3 and NMDA receptors or other synaptic proteins.

• Functional assays: Combine electrophysiology with molecular techniques to correlate DLG3 expression with synaptic plasticity measurements.

How is DLG3 being investigated in relation to neurodevelopmental disorders?

DLG3 research in neurodevelopmental disorders focuses on:

• Genetic screening: Identification of DLG3 mutations in patients with X-linked cognitive disability, such as the stop gain variant c.195del/p.(Thr66ProfsTer55) found in the MRX20 family .

• Transcriptome analysis: Differential expression studies comparing affected and unaffected individuals have identified 14 significantly differentially expressed genes between affected and unaffected males in families with DLG3 mutations .

• Pathway enrichment: Analyses have identified the "hematopoietic cell lineage" pathway as significantly enriched in DLG3 mutation carriers, with potential connections to the Serum Response Factor (SRF), an important transcription factor in the brain .

• Animal models: Development of DLG3 knockout or mutation-carrying animal models to study behavioral, electrophysiological, and molecular consequences of DLG3 dysfunction.

• Therapeutic targeting: Exploration of strategies to modulate NMDA receptor signaling to compensate for DLG3 deficiencies in neurodevelopmental disorders.

What is the significance of DLG3 downregulation in glioblastoma research?

Research has revealed that DLG3 is downregulated in glioblastoma multiforme (GBM), the most malignant form of glioma . This finding has prompted several research directions:

• Tumor suppression mechanism: Investigation of DLG3's potential role in negatively regulating cell proliferation through interaction with the adenomatosis polyposis coli tumor suppressor protein .

• Diagnostic biomarker potential: Evaluation of DLG3 expression levels as a diagnostic or prognostic marker for glioblastoma.

• Molecular classification: Integration of DLG3 expression data into molecular classification systems for brain tumors.

• Therapeutic implications: Exploration of strategies to restore DLG3 expression or function as a potential therapeutic approach for glioblastoma.

• Cell differentiation: Investigation of the relationship between DLG3 downregulation and the loss of neuronal differentiation characteristics in glioblastoma cells.

How can researchers effectively study the interaction between DLG3 and NMDA receptors?

To effectively study DLG3-NMDA receptor interactions:

• Co-immunoprecipitation (Co-IP): Use antibodies against DLG3 to pull down associated NMDA receptor subunits (particularly NR2B) and vice versa.

• Proximity ligation assay (PLA): Visualize in situ interactions between DLG3 and NMDA receptor subunits at the single-molecule level in neuronal preparations.

• FRET/BRET analysis: Employ fluorescence or bioluminescence resonance energy transfer to measure direct protein-protein interactions in living neurons.

• Domain mapping: Generate truncation constructs to identify specific domains of DLG3 responsible for NMDA receptor interaction, focusing on PDZ domains.

• Functional electrophysiology: Combine molecular manipulations of DLG3 with patch-clamp recordings to correlate protein interactions with NMDA receptor function and synaptic plasticity.