GRID2IP Antibody, FITC conjugated

What is GRID2IP and what cellular function does it serve?

GRID2IP (Glutamate receptor, ionotropic, delta 2-interacting protein 1), also known as Delphilin, functions as a postsynaptic scaffolding protein primarily localized at the parallel fiber-Purkinje cell synapse in the cerebellum. Its primary role is to serve as a molecular bridge linking the GRID2 receptor with the actin cytoskeleton and various signaling molecules . This interaction is crucial for maintaining proper synaptic architecture and signaling pathways in cerebellar circuits. The protein has been identified in human, mouse, and rat tissues, with reported reactivity across these species for most commercially available antibodies .

GRID2IP contains multiple functional domains that facilitate its scaffolding capabilities, allowing it to participate in complex molecular interactions at the postsynaptic density. Understanding the localization and expression patterns of this protein provides important insights into synaptic organization and function, particularly in cerebellum-dependent motor learning and coordination.

What are the key specifications of commercially available GRID2IP-FITC antibodies?

Currently available GRID2IP antibodies conjugated with FITC demonstrate diversity in their targeting epitopes and applications. These polyclonal antibodies are predominantly raised in rabbits against specific amino acid sequences of the human GRID2IP protein . Key specifications include:

• Host species: Rabbit

• Antibody type: Polyclonal IgG

• Conjugation: FITC (Fluorescein Isothiocyanate)

• Targeted epitopes: Various regions including AA 35-156 and AA 921-1020

• Reactivity: Primary reactivity to human GRID2IP, with cross-reactivity to mouse and rat depending on the specific product

• Applications: Immunofluorescence (paraffin-embedded and frozen sections), immunofluorescence on cultured cells, and ELISA

• Recommended dilutions: Typically 1:50-200 for immunofluorescence applications

• Storage conditions: Usually preserved in solutions containing 0.03% Proclin 300 or similar preservatives, 50% glycerol, and PBS at pH 7.4

These specifications are critical considerations when selecting the appropriate antibody for specific research applications, as they directly influence experimental outcomes and data interpretation.

What is the principle behind FITC conjugation to antibodies?

FITC conjugation involves the covalent attachment of fluorescein isothiocyanate molecules to primary amino groups on antibodies, primarily lysine residues and the N-terminal amino group . This chemical process creates a stable thiourea bond between the fluorophore and the antibody protein structure. The conjugation process is optimized for specific conditions:

• Reaction temperature, pH, and protein concentration significantly affect conjugation efficiency

• Maximal labeling is typically achieved in 30-60 minutes at room temperature

• Optimal conditions include pH 9.5 and an initial protein concentration of approximately 25 mg/ml

• The molecular fluorescein/protein (F/P) ratio is a critical parameter that determines the brightness and specificity of the conjugated antibody

When properly optimized, FITC conjugation provides direct fluorescent detection without requiring secondary antibodies, streamlining immunostaining protocols while maintaining antibody specificity and affinity . The resulting conjugates emit bright green fluorescence (emission maximum ~520 nm) when excited with blue light (excitation maximum ~495 nm), making them compatible with standard fluorescence microscopy equipment.

How should FITC-conjugated GRID2IP antibodies be stored to maintain optimal activity?

Proper storage is crucial for preserving both antibody functionality and fluorophore activity over time. The following guidelines should be observed:

• Store at 2-8°C for short-term use (up to 1 month)

• For long-term storage, aliquot and maintain at -20°C to prevent repeated freeze-thaw cycles

• Protect from light at all times as FITC is photosensitive and prone to photobleaching

• Most commercial preparations contain preservatives (0.03% Proclin 300 or 0.01% sodium azide) and stabilizers (50% glycerol in PBS at pH 7.4)

• Avoid acidic conditions, as FITC fluorescence is pH-dependent and significantly diminishes below pH 7.0

• Exercise caution when working with preservatives like Proclin, which is designated as a hazardous substance requiring appropriate handling protocols

Proper storage conditions ensure maximal antibody performance and consistent results across experiments. For antibodies used frequently, creating small working aliquots can minimize exposure to conditions that accelerate degradation.

What controls are essential when using GRID2IP-FITC antibodies in experimental protocols?

Robust experimental design requires incorporation of multiple controls to validate specificity and reliability:

• Negative tissue control: Tissues known not to express GRID2IP or tissues from GRID2IP knockout models

• Isotype control: FITC-conjugated rabbit IgG with no specific target to assess non-specific binding

• Blocking peptide control: Pre-incubating the antibody with excess immunizing peptide to confirm binding specificity

• Cross-reactivity assessment: Since some GRID2IP antibodies may have slight cross-reactivity with other proteins (such as CD166 antigen as noted in product documentation), appropriate additional controls should be included

• Autofluorescence control: Unstained sample sections to establish baseline tissue fluorescence

• Secondary antibody-only control (if using unconjugated primary antibodies in parallel experiments)

• Positive control: Tissues with confirmed GRID2IP expression (typically cerebellar sections with Purkinje cells)

These controls help discriminate between genuine GRID2IP labeling and technical artifacts, enhancing data reliability and reproducibility. Systematic documentation of control results provides crucial validation for experimental findings.

What are the recommended protocols for immunofluorescence staining using GRID2IP-FITC antibodies?

Optimal immunofluorescence protocols for GRID2IP-FITC antibodies typically follow this general workflow, with specific modifications based on sample type:

For paraffin-embedded tissue sections:

• Deparaffinize and rehydrate sections through xylene and graded alcohols

• Perform antigen retrieval (heat-induced citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Block endogenous peroxidase activity if dual immunoenzyme/immunofluorescence is planned

• Apply protein blocking solution (5-10% normal serum in PBS) for 30-60 minutes

• Incubate with GRID2IP-FITC antibody at 1:50-200 dilution overnight at 4°C or 1-2 hours at room temperature

• Wash thoroughly with PBS (3×5 minutes)

• Counterstain nuclei with DAPI if desired

• Mount with anti-fade mounting medium to preserve fluorescence

• Store slides in the dark at 4°C until imaging

For cultured cells:

• Fix cells (4% paraformaldehyde for 15 minutes or methanol for 10 minutes at -20°C)

• Permeabilize with 0.1-0.3% Triton X-100 for 10 minutes

• Block with 5% normal serum in PBS for 30-60 minutes

• Incubate with GRID2IP-FITC antibody at recommended dilution (1:50-200)

• Follow steps 6-9 as above

These protocols should be optimized for specific experimental conditions, tissue types, and research questions to achieve optimal signal-to-noise ratios.

How can researchers address weak or absent signal when using GRID2IP-FITC antibodies?

Suboptimal staining with GRID2IP-FITC antibodies can result from various factors that can be systematically addressed:

• Epitope masking: Try alternative antigen retrieval methods with varying buffer compositions, pH levels, and heating times

• Fixation issues: Different fixatives (paraformaldehyde, methanol, acetone) can differentially preserve epitopes; test multiple fixation protocols

• Antibody concentration: Use more concentrated antibody solutions by decreasing dilution factors (e.g., 1:25 instead of 1:100)

• Incubation conditions: Extend incubation time (overnight at 4°C) or adjust temperature

• Photobleaching: Minimize exposure to light during all procedural steps and use fresh anti-fade mounting medium

• Target protein expression: Verify GRID2IP expression levels in your specific sample type through complementary techniques

• Antibody quality: FITC conjugates may deteriorate over time; use fresh aliquots and verify fluorophore activity

Systematic modification of these parameters through controlled experiments can help identify and resolve specific issues affecting signal detection. Documentation of optimization steps provides valuable reference for future experiments.

What strategies help minimize background fluorescence when using FITC-conjugated antibodies?

High background can significantly obscure specific GRID2IP signals. The following approaches can improve signal-to-noise ratio:

• Optimize blocking: Extend blocking time (2+ hours) or test different blocking agents (BSA, normal serum, commercial blockers)

• Increase antibody dilution: Test more dilute antibody solutions to reduce non-specific binding

• Add washing detergent: Include 0.05-0.1% Tween-20 in wash buffers to reduce hydrophobic interactions

• Reduce autofluorescence: Treat sections with sodium borohydride or commercial autofluorescence quenching reagents

• Filter antibody solution: Centrifuge or filter antibody solution to remove aggregates that cause punctate background

• Optimize F/P ratio: Very high F/P ratios can increase background; products with moderate F/P ratios (3-6) often provide better signal-to-noise

• Consider spectral properties: Use microscope filter sets optimized for FITC to minimize bleed-through from other fluorophores

Balancing these parameters is crucial, as excessive background reduction measures can sometimes diminish specific signal intensity as well.

How does the fluorescein/protein (F/P) ratio impact experimental results with GRID2IP-FITC conjugates?

The F/P ratio, representing the average number of FITC molecules attached to each antibody molecule, significantly influences staining characteristics:

• Optimal range: Generally 3-6 FITC molecules per antibody provides balanced brightness and specificity

• Low F/P ratios (<2): Result in dim fluorescence that may be difficult to detect, especially in tissues with high autofluorescence

• High F/P ratios (>8): Can cause increased background, reduced antibody affinity, and paradoxical signal reduction through self-quenching

According to literature, maximal labeling with appropriate F/P ratios is achieved under specific conjugation conditions including 30-60 minute reaction times at room temperature, pH 9.5, and protein concentrations around 25 mg/ml . Commercial antibodies typically undergo optimization to achieve appropriate F/P ratios, but this parameter should be considered when troubleshooting unexpected results or comparing performance between different antibody lots.

How can GRID2IP-FITC antibodies be utilized in multi-labeling experiments to study synaptic organization?

Multi-labeling approaches provide valuable insights into GRID2IP's relationships with other synaptic proteins:

• Compatible fluorophores: Combine GRID2IP-FITC with antibodies conjugated to spectrally distinct fluorophores (TRITC, Cy5, Alexa 647) targeting other proteins of interest

• Sequential staining: When using multiple rabbit-derived antibodies, perform sequential immunostaining with blocking steps between applications

• Proven combinations: GRID2IP-FITC pairs effectively with markers for post-synaptic density proteins, glutamate receptors, or presynaptic terminals

• Technical considerations:

  • Include appropriate single-stained controls to assess signal bleed-through

  • Apply the FITC-conjugated antibody last when possible to minimize photobleaching

  • Utilize spectral unmixing for closely overlapping fluorophores

  • Consider antibody host species and isotypes to avoid cross-reactivity issues

These multi-labeling approaches provide spatial context for GRID2IP localization relative to functional synaptic partners, offering insights into protein interactions and synaptic architecture that cannot be obtained from single-label experiments.

What considerations are important for quantitative analysis of GRID2IP expression using FITC-conjugated antibodies?

Quantitative immunofluorescence analysis requires rigorous standardization:

• Image acquisition parameters: Maintain identical exposure settings, gain, offset, and objective magnification across all samples

• Saturation avoidance: Ensure fluorescence intensity falls within the dynamic range of the detector

• Sampling strategy: Implement systematic random sampling to avoid selection bias

• Quantifiable parameters:

  • Mean fluorescence intensity within defined regions of interest

  • Integrated density (area × mean intensity)

  • Puncta density (number of GRID2IP-positive puncta per unit area)

  • Co-localization coefficients with synaptic markers

• Software analysis: Utilize specialized image analysis software (ImageJ/FIJI, CellProfiler) with appropriate plugins

• Normalization strategies: Consider normalizing GRID2IP signal to an internal control to account for staining variability

• Statistical approach: Apply appropriate statistical tests based on data distribution and experimental design

Standardized quantitative approaches allow objective comparison of GRID2IP expression across experimental conditions, genotypes, or disease states with appropriate statistical rigor.

How can researchers validate the specificity of GRID2IP-FITC antibody staining in neuronal preparations?

Comprehensive validation strategies include:

• Genetic controls: Compare staining in wild-type versus GRID2IP knockout or knockdown models

• Epitope competition: Pre-absorb antibody with excess immunizing peptide to block specific binding sites

• Complementary detection methods: Correlate immunofluorescence findings with in situ hybridization for GRID2IP mRNA

• Multiple antibodies: Compare staining patterns using antibodies targeting different GRID2IP epitopes (e.g., AA 35-156 versus AA 921-1020)

• Recombinant expression: Test antibody on heterologous cells transfected with GRID2IP expression constructs

• Western blot correlation: Confirm antibody specificity via Western blot analysis of tissue lysates

• Pattern analysis: Verify that the observed subcellular distribution aligns with GRID2IP's known localization at parallel fiber-Purkinje cell synapses