DLG4 Antibody

What is DLG4 and why is it an important target for antibody-based research?

DLG4 (Discs large homolog 4), also known as PSD-95 or SAP-90, is a scaffolding protein of the membrane-associated guanylate kinase (MAGUK) family. It is encoded by the DLG4 gene in humans and is a critical component of the postsynaptic density in neurons. DLG4 heteromultimerizes with another MAGUK protein, DLG2, and is recruited into NMDA receptor and potassium channel clusters .

DLG4 is particularly important in neuroscience research because it:

• Forms multimeric scaffolds for clustering receptors, ion channels, and signaling proteins

• Plays a vital role in synaptic plasticity and stabilization

• Orchestrates synaptic development

• Is implicated in several neurological disorders including Huntington's disease, schizophrenia, and autism spectrum disorders

As a predominantly brain-expressed protein, DLG4 antibodies provide valuable tools for studying neuronal structure, synaptic function, and neurological disease mechanisms.

What applications are most reliable for DLG4/PSD-95 antibodies?

According to extensive validation data, DLG4 antibodies perform reliably in multiple applications with specific optimization requirements:

For optimal results in brain tissue, antigen retrieval with TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) can also be effective. The most extensively validated applications based on published literature are Western blotting (188 publications) and immunofluorescence (45 publications) .

What molecular weight should be expected when detecting DLG4 using Western blot?

When performing Western blot analysis using DLG4 antibodies, researchers should expect the following:

• Calculated molecular weight: 85 kDa

• Observed molecular weight: 90-95 kDa

• Potential additional bands: Some antibodies detect additional proteins at >100 kDa, ~75 kDa, and 50 kDa in rat and mouse samples

The discrepancy between calculated and observed molecular weights is primarily due to post-translational modifications of the protein. When validating a new DLG4 antibody, always run appropriate positive controls using brain tissue samples (mouse or rat brain tissue is recommended) .

How can I optimize DLG4 antibody staining in brain tissue sections?

For optimal DLG4/PSD-95 immunostaining in brain tissue, consider these evidence-based recommendations:

• Fixation protocol:

  • Perfusion with 4% paraformaldehyde provides optimal preservation of synaptic structures

  • Post-fixation time should be limited to 24 hours to prevent antigen masking

• Antigen retrieval methods:

  • Primary recommendation: TE buffer at pH 9.0

  • Alternative approach: Citrate buffer at pH 6.0

  • Heat-mediated retrieval (95-100°C for 15-20 minutes) shows superior results compared to enzymatic methods

• Antibody dilution optimization:

  • For immunohistochemistry: Begin with 1:500 dilution and adjust based on signal intensity

  • For immunofluorescence: Start with 1:200 dilution for most commercial antibodies

• Signal amplification considerations:

  • Tyramide signal amplification can enhance detection of low abundance synaptic proteins

  • For fluorescence applications, longer primary antibody incubation (24-48 hours at 4°C) improves signal-to-noise ratio

• Background reduction strategies:

  • Include 0.3% Triton X-100 to improve antibody penetration

  • Block with 5-10% normal serum from the same species as the secondary antibody

  • For mouse-derived antibodies on mouse tissue, use specialized mouse-on-mouse blocking reagents

These methodological approaches have been validated across multiple studies and significantly improve the quality of DLG4 immunolabeling in complex neural tissues.

What are the key considerations for detecting DLG4 in neurodevelopmental disorder models?

When investigating DLG4/PSD-95 in neurodevelopmental disorder models, researchers should consider several methodological factors:

• Animal model selection:

  • Dlg4 knockout mice exhibit behavioral phenotypes relevant to various neurodevelopmental disorders including repetitive behaviors, abnormal social behaviors, and increased anxiety-related responses

  • Dlg4-/- mice show specific deficits in motor coordination (rotarod performance) and altered stress reactivity that may confound behavioral assessments

• Experimental design concerns:

  • Age-dependent effects: DLG4 expression and localization changes significantly during development

  • Sex differences: Consider analyzing male and female animals separately as DLG4 regulation can be sexually dimorphic

  • Control selection: Littermate controls are essential due to potential compensatory mechanisms in genetic models

• Biochemical analysis approaches:

  • Subcellular fractionation is critical for accurate quantification of synaptic DLG4

  • Analysis of DLG4 ubiquitination through MDM2-mediated pathways provides insight into AMPA receptor surface expression during synaptic plasticity

  • Co-immunoprecipitation studies can reveal altered protein interactions in disease models

• Relevant molecular pathways to examine:

  • NMDA receptor clustering and function

  • Cyln2 expression (significantly reduced in Dlg4 -/- mice)

  • Expression of genes including Atp7a, Sdcbp, Sptlc2 (upregulated) and Nr4a1, Fos, Egr2, Per1, Junb, Gadd45b (downregulated)

Research has established significant associations between DLG4 gene variation and neural signatures of Williams' syndrome, particularly reduced intraparietal sulcus volume and abnormal cortico-amygdala coupling .

How can I distinguish between DLG4 and other MAGUK family proteins in my experiments?

Differentiating DLG4/PSD-95 from closely related MAGUK family proteins requires careful methodological considerations:

• Antibody selection strategies:

  • Choose antibodies raised against unique peptide sequences rather than conserved domains

  • Antibodies targeting the C-terminal region of DLG4 show higher specificity

  • Validate antibody specificity using brain tissue from Dlg4 knockout mice when possible

• Cross-reactivity assessment:

  • Most commercial DLG4 antibodies are validated against DLG2 (PSD-93) cross-reactivity

  • Testing antibodies in heterologous expression systems expressing individual MAGUK proteins provides definitive specificity data

  • Western blotting can differentiate based on molecular weight (DLG4: 90-95 kDa, DLG1: 100-130 kDa, DLG2: 80-97 kDa)

• Application-specific recommendations:

  • For immunoprecipitation: Use antibodies against non-conserved N-terminal regions

  • For immunohistochemistry: Different MAGUKs have distinct subcellular localizations that aid identification

  • For super-resolution microscopy: Dual labeling with antibodies against other synaptic markers helps confirm identity

• Technical validation approaches:

  • RNA interference to specifically deplete DLG4 confirms antibody specificity

  • Epitope competition assays with immunizing peptides verify binding specificity

  • Mass spectrometry analysis of immunoprecipitated proteins provides definitive identification

When interpreting results, consider that DLG4 and DLG2 interact at postsynaptic sites and may co-localize, potentially complicating interpretation of imaging studies .

What are common sources of variability in DLG4 antibody experiments and how can they be addressed?

Several factors contribute to variability in DLG4 antibody experiments:

• Sample preparation variables:

  • Postmortem interval significantly affects DLG4 detection in brain samples

  • Snap-freezing vs. aldehyde fixation alters epitope accessibility

  • Phosphorylation state of DLG4 changes rapidly after tissue harvesting

• Technical variability sources:

  • Freeze-thaw cycles reduce antibody activity; aliquoting is recommended for long-term storage

  • Storage buffer composition affects stability (PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 is optimal)

  • Antibody concentration varies between suppliers (0.25-0.5 mg/ml)

• Standardization approaches:

  • Use pooled reference samples across experimental batches

  • Include standard curves with recombinant DLG4 protein

  • Normalize to total protein rather than housekeeping genes for Western blots

  • For immunofluorescence, use intensity calibration beads to standardize across imaging sessions

• Recommended controls:

  • Positive control: Mouse or rat brain tissue (cerebral cortex or hippocampus)

  • Negative control: Non-neuronal tissue or Dlg4 knockout tissue

  • Peptide blocking: Pre-incubation with immunizing peptide should eliminate specific staining

For optimal reproducibility, titrate antibody in each experimental system; recommended starting dilutions vary significantly between applications (1:5000-1:50000 for WB; 1:500-1:2000 for IHC; 1:50-1:500 for IF) .

How do alternative polyadenylation events affect DLG4 antibody binding and experimental interpretation?

Research has revealed important implications of alternative polyadenylation (APA) events for DLG4 antibody-based studies:

• Impact on DLG4 transcript diversity:

  • APA generates DLG4 transcripts of varying lengths from a single gene

  • Brain tissue shows the highest frequency of APA events among all tissues for DLG4

  • The probability of APA events increases during early ischemia and under hypoxic conditions

• Implications for antibody epitope accessibility:

  • C-terminal antibodies may not detect all DLG4 isoforms if APA affects this region

  • N-terminal antibodies provide more consistent detection across APA variants

  • Antibodies targeting different regions may yield divergent results in the same sample due to APA-generated isoforms

• Methodological recommendations:

  • Use multiple antibodies targeting different DLG4 epitopes when studying stress conditions

  • Consider transcript analysis in parallel with protein detection

  • When studying hypoxic/ischemic conditions, account for potential shifts in DLG4 APA patterns

• Data interpretation considerations:

  • DLG4 expression changes may reflect altered APA patterns rather than transcriptional regulation

  • Correlate DLG4 detection with anti-apoptotic and apoptotic factor expression to interpret neuroprotective effects

  • Environmental stressors can trigger APA events that affect antibody binding characteristics

Understanding these molecular mechanisms is critical for correctly interpreting antibody-based measurements of DLG4, particularly in neuropathological conditions where APA regulation may be altered.

What are the advantages and limitations of different types of DLG4 antibodies?

Different types of DLG4 antibodies offer distinct advantages and limitations for research applications:

Species-specific considerations:

• Rabbit-origin antibodies typically show higher sensitivity for DLG4 detection in rodent samples

• Mouse monoclonals may require specialized blocking when used on mouse tissue to reduce background

• Most commercial antibodies have been validated for human, mouse, and rat reactivity

Application-specific recommendations:

• For Western blotting: Both polyclonal and monoclonal antibodies perform well

• For immunofluorescence: Polyclonal antibodies often provide superior signal in fixed tissues

• For super-resolution microscopy: High-specificity monoclonal or recombinant antibodies minimize background

• For flow cytometry: Monoclonal antibodies reduce non-specific binding

The ideal antibody selection depends on experimental goals, with recombinant technologies offering the best consistency for longitudinal studies .

How can DLG4 antibodies be used in studying the relationship between neurological disorders and synaptic dysfunction?

DLG4 antibodies have become important tools for investigating synaptic dysfunction in neurological disorders:

• Williams' syndrome research applications:

  • DLG4 antibodies can detect altered Cyln2 protein levels, which is significantly downregulated in Dlg4 -/- mice

  • Used to study abnormal dendritic spine morphology in amygdala neurons that may underlie anxiety phenotypes in Williams' syndrome

  • Help identify altered cortico-amygdala coupling associated with DLG4 SNPs

• Autism spectrum disorder investigations:

  • Detection of abnormal postsynaptic protein complexes in autism models

  • Quantification of synaptic density alterations in specific brain regions

  • Assessment of altered NMDA receptor clustering in Dlg4 knockout models showing repetitive behaviors and social interaction abnormalities

• Ischemic brain injury and neuroprotection studies:

  • Monitoring DLG4 methylation states in different brain regions following ischemia

  • Investigating fastigial nucleus stimulation (FNS) effects on DLG4 expression as a neuroprotective mechanism

  • Studying DNA methylation regulation of DLG4 in the cerebellum, which shows distinct methylation patterns compared to other brain regions

• Experimental design considerations:

  • Include region-specific analysis as DLG4 expression and function varies across brain structures

  • Consider age and developmental stage due to significant changes in DLG4 regulation during development

  • Analyze both DLG4 protein levels and post-translational modifications when assessing synaptic dysfunction

Research shows that DLG4 exhibits positive correlation with anti-apoptotic genes and negative correlation with apoptotic factors, supporting its neuroprotective role during stress responses .

What are the latest methodological advances in using DLG4 antibodies for super-resolution microscopy?

Super-resolution microscopy with DLG4 antibodies has revolutionized synaptic structure visualization:

• Optimized immunolabeling protocols:

  • Smaller probes (Fab fragments, nanobodies) provide better penetration and spatial resolution

  • Sequential labeling approaches reduce antibody crowding at dense synaptic sites

  • Heavy metal-resistant resin embedding preserves ultrastructure while maintaining antibody epitopes

• Multi-channel imaging considerations:

  • Careful antibody selection prevents bleed-through in closely related emission spectra

  • Primary antibodies from different host species enable clear differentiation between markers

  • Validated combinations include DLG4 with GluR2, Homer1, and Bassoon for pre/post-synaptic analysis

• Quantitative analysis approaches:

  • 3D object-based colocalization provides more accurate association measures than pixel-based methods

  • Machine learning algorithms improve detection of synaptic puncta in densely labeled tissue

  • Distance measurements between DLG4 and other synaptic proteins reveal organizational principles

• Technical recommendations:

  • For STORM/PALM: Use high-quality monoclonal or recombinant antibodies with minimal background

  • For STED microscopy: Optimize fixation to minimize tissue autofluorescence

  • For expansion microscopy: Test epitope preservation under expansion conditions

These advanced approaches have enabled visualization of nanoscale reorganization of DLG4 during synaptic plasticity and in disease models, providing mechanistic insights not possible with conventional microscopy.

How can epigenetic regulation of DLG4 be studied using antibody-based approaches?

Investigating epigenetic regulation of DLG4 requires specialized antibody-based strategies:

• DNA methylation analysis techniques:

  • Methylation-specific immunoprecipitation with DLG4 promoter-specific primers

  • Antibodies against methylated DNA (5-mC) combined with DLG4 locus-specific analysis

  • Studies reveal that DLG4 methylation in the cerebellum differs significantly from other brain regions, including the frontal cortex, entorhinal cortex, and superior temporal gyrus

• Chromatin immunoprecipitation (ChIP) approaches:

  • ChIP using antibodies against histone modifications at the DLG4 promoter

  • Sequential ChIP to identify transcription factor complexes regulating DLG4

  • Analysis of chromosomal regions on rat chromosomes 3, 5, 11, 15, 20, and X that correlate with DLG4 expression

• Protein-level epigenetic regulation:

  • Ubiquitination analysis of DLG4 through MDM2-mediated pathways that regulate AMPA receptor surface expression

  • Phosphorylation-specific antibodies to detect regulatory post-translational modifications

  • Mass spectrometry validation of modification sites identified by antibody-based methods

• Integrated methodological approaches:

  • Correlation of blood and brain methylation patterns for translational research

  • Combination of tissue-specific methylation analysis with gene expression data

  • Verification of methylation effects using reporter assays with methylated vs. unmethylated DLG4 regulatory regions

Recent research demonstrates that hypomethylation of DLG4 in the cerebellum may be influenced by specific sequences on multiple chromosomes, potentially upregulating neuroprotective genes .