DLG4 Antibody,HRP conjugated

What is DLG4 and why is it significant in neuroscience research?

DLG4, also known as PSD-95 (Postsynaptic density protein 95) or SAP-90 (Synapse-associated protein 90), is a critical scaffolding protein that belongs to the membrane-associated guanylate kinase (MAGUK) family. It plays an essential role in synaptic organization by heteromultimerizing with other MAGUK proteins like DLG2 and is recruited into NMDA receptor and potassium channel clusters . The protein forms multimeric scaffolds at postsynaptic sites, facilitating the clustering of receptors, ion channels, and associated signaling proteins .

DLG4 exhibits specific cellular localization patterns, being predominantly found in cell membranes, synaptic junctions, postsynaptic cell membranes, and postsynaptic densities. It shows high expression levels in the postsynaptic density of forebrain neurons and in the presynaptic regions of inhibitory synapses formed by cerebellar basket cells on axon hillocks of Purkinje cells . This distinctive distribution pattern makes DLG4 an invaluable marker for studying synaptic architecture and function in neurobiological research.

What experimental applications are suitable for HRP-conjugated DLG4 antibodies?

HRP-conjugated DLG4 antibodies are versatile tools applicable across multiple experimental platforms:

When designing experiments, researchers should consider that HRP-conjugated DLG4 antibodies typically detect bands at approximately 100 kDa, though additional cross-reactive bands may appear at >100 kDa, ~75 kDa, and 50 kDa in rat and mouse samples . These additional bands should be accounted for during experimental planning and analysis.

How do sandwich ELISA assays employ DLG4 antibodies?

Sandwich ELISA for DLG4 utilizes a systematic approach for sensitive and specific quantification:

• Anti-DLG4 antibody is pre-coated onto 96-well plates, creating a capture surface

• Biotin-conjugated anti-DLG4 antibody serves as the detection antibody

• Samples and standards are added to wells and incubated, allowing DLG4 to bind to the coated antibody

• After washing away unbound materials, the biotinylated detection antibody is added, binding to DLG4 captured by the coated antibody

• Following another wash, HRP-streptavidin conjugate is introduced to bind to the biotinylated antibodies

• After a final wash, TMB substrate is added, which is catalyzed by HRP to produce a blue color that turns yellow upon addition of stop solution

• Absorbance is measured at 450nm, with DLG4 concentration proportional to OD450 values

This method allows for precise quantification of DLG4 in various experimental samples, provided appropriate controls and standard curves are established.

How can HRP-conjugated DLG4 antibodies be utilized in investigations of protein-mRNA interactions?

Recent research has revealed complex interactions between DLG4 mRNA and RNA-binding proteins such as FMRP (Fragile X Mental Retardation Protein) and FXR1 (Fragile X-Related Protein 1). These interactions can be investigated using coimmunoprecipitation followed by detection with HRP-conjugated DLG4 antibodies.

In studies examining FXR1 binding to Dlg4 mRNA, researchers have employed deficient FMRP-N2a cells to isolate the effects of FXR1. RNA coimmunoprecipitated with anti-FXR1 antibody from these cell extracts was analyzed by qRT-PCR, revealing the presence of Dlg4 mRNA in the precipitants and confirming its association with FXR1 protein .

Methodology for protein-mRNA interaction studies with DLG4:

• Prepare cell extracts under conditions that preserve RNA-protein interactions

• Immunoprecipitate with antibodies against proteins of interest (e.g., FXR1)

• Extract RNA from immunoprecipitates

• Perform qRT-PCR to detect Dlg4 mRNA

• In parallel, use HRP-conjugated DLG4 antibodies in Western blots to confirm protein interactions

• Analyze correlation between mRNA binding and protein expression patterns

This approach provides insights into the post-transcriptional regulation of DLG4 expression and its role in synaptic function and neuronal development.

What methodological considerations apply when using DLG4 antibodies to study proteasomal interactions?

DLG4 and its associated proteins play significant roles in proteasomal degradation pathways in neurons. When investigating these interactions, several methodological considerations apply:

• Knockdown experiments: Studies have employed Dlg4-knockdown conditions to examine whether Dlg4 mRNA mediates binding of associated proteins like FXR1 to proteasomes. Results show that FXR1 coprecipitates with proteasomes even under Dlg4-knockdown conditions, suggesting that Dlg4 mRNA may not mediate FXR1 binding to proteasomes .

• Sequential immunoprecipitation: For studying complex protein interactions involving DLG4:

  • First immunoprecipitation with anti-proteasome antibodies

  • Followed by detection with HRP-conjugated DLG4 antibodies

  • Further immunoprecipitation with antibodies against other proteins of interest (FXR1, FMRP)

  • Analysis of precipitates for both protein content and associated mRNAs

• Controls: Include appropriate controls such as IgG immunoprecipitation and RNase treatment to distinguish RNA-dependent from RNA-independent interactions.

This multifaceted approach allows researchers to dissect the complex relationship between DLG4, its mRNA, associated RNA-binding proteins, and the proteasomal degradation pathway in neuronal function.

How can DLG4 antibodies contribute to mapping neuronal circuit alterations in pathological conditions?

HRP-conjugated DLG4 antibodies serve as powerful tools for mapping synaptic alterations in various neurological conditions:

• Brain injury models:

  • DLG4 antibodies have been validated in rat cerebral infarction models to assess post-injury synaptic reorganization

  • Immunohistochemistry with HRP-conjugated antibodies enables visualization of altered synaptic architecture in affected regions

• Methodological workflow for neuronal circuit mapping:

  • Prepare brain sections from experimental and control animals

  • Perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

  • Block tissue sections with 10% goat serum

  • Incubate with primary anti-DLG4 antibody at appropriate concentration (e.g., 2 μg/ml) overnight at 4°C

  • Apply HRP-conjugated secondary antibody and incubate (typically 30 minutes at 37°C)

  • Develop with DAB chromogen and analyze synaptic architecture alterations

• Quantitative analysis:

  • Measure intensity and distribution of DLG4 immunoreactivity

  • Assess colocalization with other synaptic markers

  • Compare alterations across brain regions and experimental conditions

This methodology provides valuable insights into synaptic remodeling processes occurring during neurological disorders and following therapeutic interventions.

What are the optimal protocols for Western blotting using HRP-conjugated DLG4 antibodies?

For optimal Western blot results with DLG4 antibodies, researchers should follow this validated protocol:

• Sample preparation:

  • Prepare tissue lysates from relevant brain regions (rat/mouse brain or human cell lines)

  • Load approximately 30 μg of protein per lane under reducing conditions

• Electrophoresis conditions:

  • Use 5-20% SDS-PAGE gradient gels

  • Run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

• Protein transfer:

  • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat milk/TBS for 1.5 hours at room temperature

  • Incubate with anti-DLG4 antibody at appropriate concentration (e.g., 0.5 μg/mL) overnight at 4°C

  • Wash with TBS-0.1% Tween (3 times, 5 minutes each)

  • Incubate with HRP-conjugated secondary antibody at 1:5000 dilution for 1.5 hours at room temperature

• Detection:

  • Develop using enhanced chemiluminescence (ECL) kit

  • Expect to detect DLG4 band at approximately 90-100 kDa

  • Note that additional bands may appear at >100 kDa, ~75 kDa, and 50 kDa in rat and mouse samples

This protocol consistently yields specific detection of DLG4 in various neural tissues and cell types, enabling quantitative analysis of protein expression levels.

How should researchers optimize immunohistochemistry experiments with HRP-conjugated DLG4 antibodies?

Successful immunohistochemical detection of DLG4 requires careful attention to multiple experimental parameters:

• Tissue preparation:

  • Fix tissues appropriately (typically paraformaldehyde)

  • Prepare paraffin-embedded or frozen sections depending on experimental needs

• Antigen retrieval:

  • Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is essential for optimal epitope exposure

  • This step is critical as DLG4 epitopes may be masked during fixation

• Blocking and antibody incubation:

  • Block tissue sections with 10% goat serum to reduce non-specific binding

  • Incubate with primary anti-DLG4 antibody at 2 μg/ml overnight at 4°C

  • For directly HRP-conjugated antibodies, proceed directly to development

  • For unconjugated primaries, apply appropriate HRP-conjugated secondary antibody and incubate for 30 minutes at 37°C

• Signal development:

  • Develop using DAB (3,3'-diaminobenzidine) chromogen

  • Monitor reaction to achieve optimal signal-to-noise ratio

  • Counterstain as needed for structural context

• Controls and validation:

  • Include negative controls (omitting primary antibody)

  • Consider using brain tissue from DLG4-knockout models when available

  • Compare staining patterns with published literature

Following this methodological approach ensures specific and reproducible detection of DLG4 in various neural tissues, enabling accurate analysis of its distribution in physiological and pathological conditions.

What critical factors influence the storage and handling of HRP-conjugated DLG4 antibodies?

Maintaining antibody integrity is crucial for experimental success. Key considerations include:

• Storage conditions:

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

  • Typical shelf-life is 12 months under recommended storage conditions

  • Avoid repeated freeze/thaw cycles as they degrade antibody quality and HRP activity

• Shipping and receipt handling:

  • Typically shipped with ice packs

  • Upon receipt, store immediately at recommended temperature

  • Avoid prolonged exposure to room temperature

• Buffer composition effects:

  • Optimal buffer is typically phosphate buffered solution, pH 7.4

  • Inclusion of stabilizers (0.05%), protein protectants (1%), and glycerol (50%) helps maintain antibody function

  • Be aware of buffer components when designing experiments to avoid interference

• Working solution preparation:

  • Prepare fresh dilutions for each experiment

  • Use appropriate diluents compatible with the application

  • For Western blotting, typical dilutions range from 1:500-1:1000

  • For immunohistochemistry, optimal dilutions are typically 1:100-1:200

• HRP activity preservation:

  • Avoid exposure to strong oxidizing agents

  • Protect from direct light when possible

  • Consider adding sodium azide only to storage buffers, not working solutions (as it inhibits HRP)

Proper handling according to these guidelines ensures maximum sensitivity and specificity in experimental applications.

How should researchers interpret multiple band patterns in DLG4 Western blots?

Multiple band patterns in DLG4 Western blots are common and require careful interpretation:

• Expected band pattern:

  • Primary DLG4 band typically appears at ~95-100 kDa

  • Additional bands may appear at >100 kDa, ~75 kDa, and 50 kDa in rat and mouse samples

• Factors influencing band patterns:

  • Post-translational modifications: Different phosphorylation states can alter protein mobility

  • Alternative splicing: Multiple transcript variants encoding different isoforms exist for DLG4

  • Protein degradation: Partial degradation during sample preparation can generate fragments

  • Cross-reactivity: Some antibodies may detect other MAGUK family members

• Methodological approach to band verification:

  • Compare observed patterns with literature-reported patterns

  • Use tissue-specific positive controls (e.g., brain lysates) alongside experimental samples

  • Consider performing peptide competition assays to verify specificity

  • When possible, compare results using antibodies targeting different epitopes of DLG4

• Analytical considerations:

  • For quantitative analysis, clearly define which band(s) represent the target protein

  • Document all bands observed and their relative intensities

  • Maintain consistent analysis methods across experimental groups

What strategies address non-specific binding issues with HRP-conjugated DLG4 antibodies?

Non-specific binding can complicate data interpretation. These strategies can minimize such issues:

• Optimized blocking protocols:

  • Use 5% non-fat milk in TBS for Western blotting applications

  • For immunohistochemistry, 10% goat serum effectively reduces background

  • Consider species-matched normal serum based on secondary antibody source

• Antibody dilution optimization:

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

  • For Western blotting, 0.5 μg/mL has been validated for specific detection

  • For immunohistochemistry, 2 μg/ml typically provides optimal results

• Wash optimization:

  • For Western blotting, use TBS-0.1% Tween with three 5-minute washes

  • For immunohistochemistry, phosphate-buffered saline with appropriate detergent

  • Increasing wash duration/frequency can reduce non-specific binding

• Sample-specific considerations:

  • For brain tissue, consider region-specific autofluorescence/background

  • Pre-adsorption with non-relevant tissues may reduce cross-reactivity

  • Inclusion of appropriate reducing agents during sample preparation minimizes non-specific disulfide bonds

• Validation approaches:

  • Verify specificity using multiple antibody dilutions

  • Compare staining patterns across different antibodies targeting DLG4

  • Include negative controls omitting primary antibody

These methodological refinements minimize non-specific binding while preserving specific DLG4 detection, resulting in cleaner data and more reliable interpretation.

How can researchers validate DLG4 antibody specificity across different neural tissues?

Comprehensive validation ensures reliable results across diverse neural tissues:

• Cross-species reactivity assessment:

  • DLG4 antibodies have been validated in multiple species including rat, mouse, human, and cow

  • Verify antibody reactivity to your species of interest before extensive experiments

• Tissue-specific validation:

  • For brain tissue, validation has been performed in:

    • Mouse brain tissue

    • Rat brain tissue

    • Rat cerebral infarction models

    • Human U-87MG glioblastoma cells

• Methodological validation across techniques:

  • Western blotting: Verify band size and pattern consistency

  • Immunohistochemistry: Compare staining patterns with known DLG4 distribution

  • Immunofluorescence: Assess colocalization with other synaptic markers

• Knockout/knockdown validation:

  • When available, use DLG4 knockout tissues as negative controls

  • Alternatively, siRNA-mediated knockdown can provide validation controls

  • Compare antibody reactivity before and after knockdown

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Observe elimination of specific signals

  • Non-specific binding will typically remain

This multi-faceted validation approach ensures reliable and reproducible results across different neural tissues and experimental conditions.

How are HRP-conjugated DLG4 antibodies being employed in RNA-protein interaction studies?

Recent advances highlight the importance of DLG4 in complex RNA-protein interactions:

• Investigation of mRNA binding proteins:

  • DLG4 mRNA associates with RNA-binding proteins including FMRP and FXR1

  • HRP-conjugated DLG4 antibodies enable visualization of these complexes in cellular contexts

• Methodological workflow:

  • RNA immunoprecipitation followed by qRT-PCR for DLG4 mRNA

  • Parallel protein immunoprecipitation with detection via HRP-conjugated antibodies

  • Correlation of mRNA binding with protein expression and localization patterns

• Functional implications:

  • These techniques have revealed that DLG4 mRNA association with RNA-binding proteins influences synaptic plasticity

  • Understanding of post-transcriptional regulation of DLG4 expression is enhanced through these approaches

• Technical considerations:

  • RNase-free conditions throughout experimental workflow

  • Appropriate controls including RNase treatment and IgG immunoprecipitation

  • Quantitative analysis of both protein and mRNA levels

This emerging research area is expanding our understanding of the complex regulatory mechanisms governing DLG4 expression and function in neuronal systems.

What is the role of DLG4 in proteasomal degradation pathways?

Emerging research has revealed connections between DLG4 and protein degradation mechanisms:

• Protein-protein interactions:

  • FXR1 protein (which binds DLG4 mRNA) also associates with proteasomes

  • This association persists even under DLG4-knockdown conditions

  • HRP-conjugated DLG4 antibodies help visualize these interactions through Western blotting and immunoprecipitation

• Experimental approach:

  • Knockdown of DLG4 using siRNA or shRNA

  • Immunoprecipitation with anti-proteasome antibodies

  • Detection of associated proteins using HRP-conjugated antibodies

  • Analysis of effects on protein degradation pathways

• Methodological considerations:

  • Requires carefully optimized lysis conditions to preserve protein-protein interactions

  • Controls must include both knockdown verification and immunoprecipitation specificity

  • Quantitative analysis of protein levels and degradation rates

These findings suggest complex regulatory mechanisms involving DLG4, RNA-binding proteins, and proteasomal pathways in neuronal function, with HRP-conjugated antibodies serving as essential tools for their investigation.