DLG4 Antibody，FITC conjugated

What is DLG4/PSD-95 and why is it important in neuroscience research?

DLG4 (Discs Large Homolog 4), commonly known as PSD-95 (Postsynaptic Density Protein 95) or SAP-90 (Synapse-Associated Protein 90), is a critical scaffold protein in neuronal synapses. It belongs to the membrane-associated guanylate kinase (MAGUK) family with a calculated molecular weight of approximately 95 kDa .

DLG4/PSD-95 is primarily localized in the postsynaptic density of neurons in the forebrain, though it can also be found in the presynaptic region of inhibitory synapses formed by cerebellar basket cells on Purkinje cell axon hillocks . The protein heteromultimerizes with another MAGUK protein, DLG2, and is recruited into NMDA receptor and potassium channel clusters, forming a multimeric scaffold for the clustering of receptors, ion channels, and associated signaling proteins .

Its importance in neuroscience research stems from its central role in:

• Synaptic plasticity mechanisms underlying learning and memory

• Organization of postsynaptic signaling complexes

• Regulation of glutamate receptor trafficking and function

• Involvement in neurodevelopmental and neurodegenerative disorders

How do I distinguish between various clones and formats of FITC-conjugated DLG4 antibodies?

When selecting FITC-conjugated DLG4 antibodies, researchers must consider multiple factors based on their experimental needs:

For example, clone 7E3 (ABIN2484833) is a mouse monoclonal IgG1 antibody that reacts with rat DLG4 and is suitable for WB, IHC, IF, ICC, and antibody array applications . In contrast, there are also phospho-specific antibodies like ABIN745005, which specifically recognizes DLG4 phosphorylated at Tyr236 and Tyr240 residues .

What experimental applications are compatible with FITC-conjugated DLG4 antibodies?

FITC-conjugated DLG4 antibodies can be utilized in various experimental applications:

Most FITC-conjugated DLG4 antibodies have an excitation/emission profile of 499/515 nm and are compatible with 488 nm laser lines in confocal microscopy and flow cytometry applications .

How can I optimize staining protocols when using FITC-conjugated DLG4 antibodies for immunofluorescence?

Optimizing protocols for FITC-conjugated DLG4 antibodies requires attention to several key parameters:

Sample preparation:

• For brain tissue, use heat-mediated antigen retrieval in EDTA buffer (pH 8.0) for optimal epitope accessibility

• For cultured neurons, 4% paraformaldehyde fixation for 15-20 minutes typically preserves DLG4 epitopes well

Protocol optimization:

• Block with 10% serum (matching the host species of secondary antibody) to reduce background

• For paraffin-embedded sections, use 5 μg/mL antibody concentration overnight at 4°C

• Since DLG4 is primarily localized at synapses, use confocal microscopy to visualize the punctate staining pattern

Specific considerations for FITC conjugates:

• FITC is sensitive to photobleaching, so minimize exposure to light during all steps

• Mount slides with anti-fade mounting medium containing DAPI for nuclear counterstaining

• For better signal preservation, store slides at 4°C in the dark after mounting

Successful staining should reveal punctate labeling of DLG4/PSD-95 at synaptic sites, particularly in the forebrain regions, with minimal background in other cellular compartments .

What are the most effective controls when using FITC-conjugated DLG4 antibodies?

Implementing proper controls is crucial for validating experimental results with FITC-conjugated DLG4 antibodies:

For mouse monoclonal antibodies like clones 6G6 (IgG2a) or 7E3 (IgG1), matching isotype controls should be used . When using FITC-conjugated antibodies, include an unstained sample to establish autofluorescence levels in your specific tissue or cell type.

How do I troubleshoot weak or non-specific signals when using FITC-conjugated DLG4 antibodies?

Common issues with FITC-conjugated DLG4 antibodies and their solutions:

Weak signal:

• Increase antibody concentration (try 2-5 μg/mL range)

• Extend incubation time (overnight at 4°C instead of 1-2 hours)

• Optimize antigen retrieval method (EDTA buffer pH 8.0 works well for brain tissue)

• Ensure samples were properly fixed and permeabilized

• Check fluorescence microscope settings (FITC excitation: 499 nm, emission: 515 nm)

High background/non-specific signal:

• Increase blocking time or concentration (10% serum is recommended)

• Reduce antibody concentration

• Include 0.1% Triton X-100 in wash buffers

• Be aware that DLG4 antibodies may detect additional cross-reactive bands at ~75 kDa and 50 kDa in rat and mouse samples

Inconsistent results:

• Ensure proper storage of antibody (at -20°C, avoid freeze/thaw cycles)

• Verify that your sample preparation preserves the epitope (some fixatives may mask the epitope)

• Consider that FITC is sensitive to photobleaching; minimize light exposure during all steps

How can I quantitatively analyze DLG4 expression or localization changes in synaptic plasticity studies?

Quantitative analysis of DLG4/PSD-95 in synaptic plasticity research requires sophisticated approaches:

For immunofluorescence image analysis:

• Acquire z-stack images using confocal microscopy with consistent settings

• Use deconvolution algorithms to improve signal-to-noise ratio

• Analyze puncta density, size, and intensity using ImageJ/FIJI with plugins like:

  • Puncta Analyzer

  • SynPAnal

  • SynD

For biochemical fractionation and analysis:

• Isolate postsynaptic density fractions using differential centrifugation

• Compare DLG4 levels in total lysate vs. synaptosomal vs. PSD fractions

• Quantify changes using Western blotting with the unconjugated version of the same antibody clone

For super-resolution approaches:

• Use FITC-conjugated DLG4 antibodies compatible with techniques like STED or STORM

• Apply cluster analysis algorithms to quantify nanoscale organization changes

• Co-localize with other synaptic markers (e.g., presynaptic or glutamate receptor proteins)

When analyzing phosphorylation-dependent changes, phospho-specific antibodies like those targeting pTyr236/pTyr240 should be used to detect activation-dependent modifications of DLG4/PSD-95 .

What are the considerations for differentiating between specific DLG4 phosphorylation states?

Phosphorylation of DLG4/PSD-95 is a critical regulatory mechanism affecting its function and protein interactions:

Key phosphorylation sites:

• Tyrosine 236/240: Important regulatory sites detected by phospho-specific antibodies like ABIN745005

• Serine/Threonine sites: Regulated by kinases including CaMKII, CDK5, and GSK3β

Experimental approach:

• Use phospho-specific antibodies like those targeting pTyr236/pTyr240

• Include appropriate controls:

  • Phosphatase treatment to verify phosphorylation specificity

  • Kinase inhibitors or activators to modulate phosphorylation state

  • Phospho-mimetic or phospho-dead mutants in expression systems

Technical considerations:

• Phospho-epitopes are labile; use phosphatase inhibitors in all buffers

• For IF applications, phospho-specific antibodies often require more stringent validation

• Some phosphorylation events may affect antibody accessibility to epitopes

The KLH-conjugated synthetic phosphopeptide derived from human PSD95 around the phosphorylation sites Tyr236/Tyr240 can be used as a blocking peptide to verify the specificity of phospho-specific antibody binding .

How can I validate the specificity of FITC-conjugated DLG4 antibodies in genetic models?

Rigorous validation of DLG4 antibodies in genetic models is essential for ensuring experimental reproducibility:

Validation in knockout/knockdown models:

• Generate or obtain DLG4/PSD-95 knockout animals or knockdown cell lines

• Perform side-by-side comparison of wildtype and knockout/knockdown samples

• Verify complete absence of signal in knockout or significant reduction in knockdown samples

• Be aware that some DLG4 antibodies may cross-react with related family members (other DLG/MAGUK proteins)

Specificity testing methods:

• Western blot: Should show absence of the ~95 kDa band in knockout samples

• Immunofluorescence: Should show absence of characteristic punctate synaptic staining

• Flow cytometry: Should show shift in fluorescence intensity distribution

Alternative validation approaches:

• Overexpression systems with tagged DLG4 constructs

• Peptide competition assays using the immunizing peptide

• Comparison of staining patterns across multiple antibodies targeting different DLG4 epitopes

When validating monoclonal antibodies like clones 6G6 or 7E3, be aware that they detect ~100 kDa band with additional cross-reactive bands at ~75 kDa and 50 kDa in rat and mouse samples .

What methodological approaches can resolve contradictory findings when using different DLG4 antibodies?

Resolving contradictory findings with different DLG4 antibodies requires systematic troubleshooting:

Sources of discrepancies:

• Epitope specificity: Different antibodies recognize distinct regions of DLG4

• Isoform detection: Multiple transcript variants of DLG4 exist

• Post-translational modifications: Phosphorylation or other modifications may mask epitopes

• Methodology differences: Fixation, antigen retrieval, or detection systems can affect results

Resolution strategies:

• Epitope mapping:

  • Determine exactly which region each antibody recognizes

  • Use epitope-tagged constructs to validate binding specificity

• Multiple antibody validation:

  • Test multiple antibodies targeting different DLG4 epitopes

  • Compare monoclonal (e.g., 6G6, 7E3) vs. polyclonal antibodies

  • Include phospho-specific antibodies if post-translational modifications are suspected

• Complementary techniques:

  • Combine imaging with biochemical approaches

  • Use proximity ligation assays to verify protein interactions

  • Employ mass spectrometry for unbiased identification

• Control experiments:

  • Use genetic models (knockout/knockdown)

  • Perform peptide competition assays

  • Include appropriate isotype controls

When comparing results across studies, note the specific antibody clone, host species, and experimental conditions used, as these factors significantly impact experimental outcomes and reproducibility.