PSD Antibody, FITC conjugated

What is PSD-95 and why is it an important target for neurobiological research?

PSD-95 (Postsynaptic Density Protein 95) is a critical scaffolding protein located predominantly in the postsynaptic density of excitatory synapses. It belongs to the membrane-associated guanylate kinase (MAGUK) family and plays essential roles in synaptic organization, trafficking, and function of neurotransmitter receptors, particularly NMDA receptors. PSD-95 serves as a crucial molecular scaffold that facilitates signal transduction complexes at synapses through its multiple protein interaction domains, including three PDZ domains, an SH3 domain, and a guanylate kinase-like domain .

The protein is also known by several alternate names including SAP90 (Synapse-associated protein 90), DLG4 (discs large homolog 4), and various combinations of these terminologies . As a key organizer of the postsynaptic machinery, PSD-95 represents an important target for investigating synaptic plasticity, neurodevelopment, and neurological disorders.

What are the species cross-reactivities for commercial PSD-95 antibodies?

Most commercially available PSD-95 antibodies, including the FITC-conjugated monoclonal antibody 7E3, demonstrate consistent cross-reactivity across multiple mammalian species. Specifically, these antibodies reliably detect PSD-95 in human, mouse, rat, and bovine samples . This broad species cross-reactivity makes these antibodies versatile tools for comparative studies across various model organisms.

Researchers should note that the expression levels of PSD-95 vary across different brain regions, with higher expression typically observed in the thalamus and cerebral cortex compared to the cerebellum and hippocampus . This regional variation should be considered when designing experiments and interpreting results.

What is the molecular weight profile of PSD-95 protein when detected by antibodies?

• An additional protein band of >100 kDa is often detected

• Cross-reactive bands at approximately 75-80 kDa in rat and mouse samples

• Cross-reactive bands at approximately 50 kDa in rat and mouse samples

These additional bands may represent alternative splice variants, post-translationally modified forms, degradation products, or cross-reactivity with related proteins. Researchers should validate their specific antibody lot against appropriate positive and negative controls to confirm specificity and to properly interpret observed banding patterns in their experimental system.

How should PSD-95 FITC-conjugated antibodies be stored to maintain optimal activity?

Proper storage of FITC-conjugated antibodies is crucial for maintaining their fluorescence intensity and binding efficiency. The following storage protocols are recommended:

• For continuous use (up to one week): Store undiluted antibody at 2-8°C in the dark

• For long-term storage: Aliquot and store at -20°C

• Avoid storage in frost-free freezers due to temperature fluctuations

• Keep the antibody protected from prolonged exposure to light to prevent photobleaching of the FITC fluorophore

• Avoid repeated freeze/thaw cycles as they can degrade antibody quality

• Prior to use, gently mix the antibody solution (avoid vortexing which can cause protein denaturation)

It is advisable to spin the vial prior to opening to collect the solution at the bottom of the tube, especially after shipment or storage . Always store FITC-conjugated antibodies in amber tubes or wrapped in aluminum foil to minimize light exposure.

What buffer compositions are optimal for PSD-95 antibody applications?

PSD-95 antibodies are typically formulated in specific buffer compositions to maintain stability and activity. Common buffer formulations include:

• PBS (phosphate-buffered saline) at pH 7.4 as the base buffer

• 50% Glycerol as a cryoprotectant for freeze storage

• 0.05-0.09% Sodium azide as a preservative to prevent microbial contamination

For specific applications like immunoprecipitation, researchers should note that a buffer containing 1% NP-40–0.5% sodium deoxycholate (DOC) mixture has been shown to effectively solubilize PSD-95 and associated proteins like NMDA receptor subunits . This detergent mixture enables successful co-immunoprecipitation studies while preserving protein-protein interactions.

When designing experiments involving membrane fractions or postsynaptic density isolation, specialized extraction buffers may be necessary to maintain the integrity of the protein complexes.

What validated applications are suitable for FITC-conjugated PSD-95 antibodies?

FITC-conjugated PSD-95 antibodies have been validated for multiple experimental applications, primarily focused on localization and visualization studies:

• Immunohistochemistry (IHC) - both frozen and paraffin-embedded sections

• Immunocytochemistry (ICC)

• Immunofluorescence (IF)

• ELISA (for some antibody products)

The 7E3 clone in particular has been extensively validated for these applications across multiple species samples . Researchers should be aware that not all antibodies may perform equally well across all applications, and optimization may be necessary for each specific experimental setup.

How can PSD-95 antibodies be used to investigate protein-protein interactions at the synapse?

PSD-95 antibodies can be employed in several sophisticated techniques to investigate protein-protein interactions:

Co-immunoprecipitation (Co-IP): Using appropriate solubilization conditions (such as 1% NP-40–0.5% DOC buffer), PSD-95 antibodies can effectively precipitate PSD-95 along with its interacting partners . Research has demonstrated the ability to co-precipitate NMDA receptor subunits (NR1 and NR2A/B) with PSD-95, confirming their physical association in the postsynaptic density .

For more sensitive detection, researchers can combine immunoprecipitation with Western blotting or mass spectrometry to identify novel binding partners. When designing these experiments, it's critical to select antibodies that don't interfere with protein interaction domains. Monoclonal antibodies like 7E3 that recognize specific epitopes may be particularly useful for such studies.

Proximity ligation assays (PLA) can be combined with FITC-conjugated PSD-95 antibodies to visualize protein interactions in situ with high specificity and sensitivity.

What strategies can minimize interference when studying PSD-95 and its binding partners simultaneously?

Recent advances have led to the development of specialized recombinant binders (such as Xph monobodies) that can bind to PSD-95 without interfering with its ability to interact with natural ligands through its PDZ domains . These engineered binders offer several advantages:

• They allow simultaneous binding of both PDZ domain ligands and the monobody

• Nuclear magnetic resonance (NMR) studies confirm that PDZ domain-binding properties remain unaltered in the presence of these binders

• Fluorescence polarization assays demonstrate that binding affinities of PSD-95 PDZ domain ligands are maintained even when the monobodies are present

For researchers investigating complex interaction networks involving PSD-95, combining these non-interfering binders with FITC-conjugated antibodies targeting other proteins can provide a powerful approach for multiplexed imaging and analysis of multi-protein complexes.

How do PSD-95 expression patterns vary across brain regions and developmental stages?

Western blot analyses have revealed significant regional variation in PSD-95 expression across different brain structures. The highest expression levels are typically observed in the thalamus and cerebral cortex, while expression is notably lower in the cerebellum and hippocampus . This regional heterogeneity has important implications for experimental design and data interpretation.

When designing immunohistochemistry or immunofluorescence experiments using FITC-conjugated PSD-95 antibodies, researchers should:

• Include appropriate positive control regions known to express high levels of PSD-95

• Adjust exposure settings to account for regional variation

• Consider using quantitative image analysis methods to accurately compare expression across regions

• Include developmental timepoints when studying synaptic maturation processes

Understanding these expression patterns is critical for studies investigating region-specific synaptic organization and function in neurological disorders.

What are common technical issues when using FITC-conjugated antibodies for immunofluorescence, and how can they be addressed?

Several technical challenges may arise when working with FITC-conjugated PSD-95 antibodies:

Photobleaching: FITC is particularly susceptible to photobleaching compared to other fluorophores.

• Solution: Minimize exposure to light during all steps

• Use anti-fade mounting media containing DABCO or propyl gallate

• Consider capturing images of FITC channels first during multi-channel imaging

Autofluorescence: Brain tissue often exhibits significant autofluorescence in the green spectrum where FITC emits.

• Solution: Include appropriate negative controls

• Use spectral unmixing during image acquisition if available

• Consider Sudan Black B treatment to reduce lipofuscin autofluorescence

• Employ appropriate background subtraction methods during image analysis

Cross-reactivity: PSD-95 antibodies may detect additional bands as noted previously.

• Solution: Validate specificity with appropriate knockout/knockdown controls

• Perform absorption controls with recombinant PSD-95 protein

• Consider using alternative clones if cross-reactivity is problematic

Signal amplification: FITC typically provides moderate brightness which may be insufficient for detecting low abundance proteins.

• Solution: Consider tyramide signal amplification methods

• Use high-sensitivity detection systems

• Optimize antigen retrieval methods for fixed tissues

What considerations are important when designing multicolor immunofluorescence experiments that include FITC-conjugated PSD-95 antibodies?

When incorporating FITC-conjugated PSD-95 antibodies into multicolor immunofluorescence experiments, several factors should be considered:

Spectral compatibility: FITC (excitation ~495 nm, emission ~520 nm) has potential spectral overlap with other green fluorophores and yellow fluorophores.

• Recommendation: Pair with far-red (Cy5, Alexa 647) and blue (DAPI) or red (Cy3, Alexa 555) fluorophores

• If using GFP-expressing tissues, consider using a different fluorophore for PSD-95 detection

Antibody cross-reactivity: When combining multiple primary antibodies from the same host species, cross-reactivity can occur with secondary antibodies.

• Recommendation: Use directly conjugated primary antibodies like the FITC-PSD-95

• Employ sequential staining protocols with intermediate blocking steps

• Consider the use of Fab fragments to block exposed IgG epitopes between staining steps

Fixation compatibility: Different epitopes may require different fixation conditions for optimal preservation.

• Recommendation: Validate each antibody individually before combining

• Test different fixation protocols (4% PFA, methanol, or combinations)

• Optimize antigen retrieval conditions for each target protein

Quantitative analysis: When quantifying colocalization, be mindful that FITC signal can bleed into other channels.

• Recommendation: Perform rigorous single-fluorophore controls

• Use appropriate background subtraction and threshold setting

• Consider employing specialized colocalization analysis software

How should researchers interpret differences in PSD-95 localization patterns across experimental conditions?

When analyzing PSD-95 localization using FITC-conjugated antibodies, researchers should consider multiple factors that influence interpretation:

Subcellular distribution: Under normal conditions, PSD-95 exhibits a punctate distribution corresponding to postsynaptic sites. Changes in this pattern may indicate:

• Synaptic reorganization or plasticity

• Pathological conditions affecting synaptic integrity

• Developmental changes in synaptic maturation

Quantitative parameters: Several quantitative measures should be considered:

• Puncta density (number of PSD-95-positive puncta per unit area)

• Puncta size (area or diameter of individual puncta)

• Fluorescence intensity (relative abundance of PSD-95)

• Colocalization with pre- or postsynaptic markers

Statistical approaches: Appropriate statistical methods should include:

• Analysis of multiple fields from multiple samples

• Normalization to appropriate housekeeping proteins

• Blinded analysis to prevent bias

• Consideration of hierarchical data structure (multiple measurements from individual animals)

Technical considerations: Variations in fixation, antibody penetration, and imaging parameters can significantly impact observed patterns and should be strictly controlled across experimental groups.

What are the most effective methods for quantifying PSD-95 clusters in immunofluorescence images?

Quantification of PSD-95 immunoreactive clusters requires rigorous methodological approaches:

Image acquisition parameters:

• Use identical exposure settings across all experimental groups

• Capture images at optimal resolution (Nyquist sampling)

• Avoid saturation by adjusting exposure times

• Acquire z-stacks to capture the full three-dimensional structure

Preprocessing steps:

• Background subtraction using rolling ball algorithm

• Deconvolution to improve signal-to-noise ratio

• Thresholding to separate signal from background

Analysis approaches:

• Automated puncta detection using specialized software (ImageJ, CellProfiler)

• 3D object counting for volumetric analysis

• Watershed segmentation for separating adjacent puncta

• Distance-based colocalization analysis with presynaptic markers

Validation considerations:

• Compare multiple thresholding methods to ensure robustness

• Validate automated counts against manual counts in a subset of images

• Include appropriate positive and negative controls

• Consider the use of computational approaches like machine learning for complex pattern recognition

By employing these systematic approaches, researchers can obtain reliable quantitative data on PSD-95 cluster properties across experimental conditions.