GRIN1 Antibody, FITC conjugated

What is GRIN1 and why is it an important research target?

GRIN1 (Glutamate receptor ionotropic, NMDA 1) is the gene encoding the GluN1 subunit, which is an obligatory component of N-methyl-D-aspartate receptors (NMDARs). These receptors are heteromeric protein complexes that form ligand-gated ion channels. The GluN1 subunit plays a critical role in the plasticity of synapses, which underlies memory and learning processes . Because of its essential function in NMDAR assembly and function, GRIN1/GluN1 is a key target for neuroscience research investigating synaptic transmission, plasticity, and neurological disorders.

What are the key characteristics of GRIN1 Antibody, FITC conjugated?

GRIN1 Antibody, FITC conjugated is a polyclonal antibody derived from rabbit hosts that recognizes the human Glutamate receptor ionotropic, NMDA 1 protein . The antibody targets a recombinant human GRIN1 protein fragment (amino acids 274-451) . It has been purified using Protein G chromatography to >95% purity and is conjugated with fluorescein isothiocyanate (FITC), which provides direct fluorescent detection capabilities . This antibody is specific for GRIN1 (also known as NMDAR1, GluN1, or Glutamate NMDA receptor subunit zeta-1) and has confirmed reactivity with human samples, though some variants also react with mouse and rat samples .

How does FITC conjugation work in antibodies?

FITC conjugation involves the attachment of fluorescein isothiocyanate to proteins through amine groups. The isothiocyanate functional group (-N=C=S) of FITC reacts with primary amines present in proteins at lysine residues and at the amino terminus . This chemical reaction creates a stable thiourea bond between the fluorophore and the antibody. The resulting FITC-conjugated antibody emits green fluorescence when excited with appropriate wavelengths (typically around 495 nm), allowing direct visualization without the need for secondary antibodies in techniques such as immunofluorescence, flow cytometry, and immunohistochemistry .

What are the optimal storage conditions and handling practices for GRIN1 Antibody, FITC conjugated?

For optimal preservation of activity, GRIN1 Antibody, FITC conjugated should be stored at -20°C or -80°C immediately upon receipt . Repeated freeze-thaw cycles should be avoided to prevent degradation of the antibody and loss of fluorescent signal. The antibody is typically supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . When working with the antibody, it's advisable to:

• Aliquot the antibody into single-use volumes before freezing

• Keep the antibody protected from light during handling and storage

• Centrifuge briefly before opening the tube to ensure all liquid is at the bottom

• Maintain cold chain conditions when removing from storage

• Return unused portions to -20°C promptly

What are the recommended dilutions for different applications using GRIN1 Antibody, FITC conjugated?

The optimal dilution varies depending on the specific application and experimental conditions. Based on the available data:

While these guidelines provide a starting point, it's strongly recommended to perform titration experiments to determine the optimal concentration for your specific experimental setup. Factors affecting optimal dilution include tissue type, fixation method, antigen abundance, and detection system sensitivity.

How should I design controls for experiments using GRIN1 Antibody, FITC conjugated?

Proper controls are essential for validating results obtained with GRIN1 Antibody, FITC conjugated:

• Positive control: Include tissues or cells known to express GRIN1, such as brain tissue from humans, mice, or rats . Cerebellum tissue has been validated for this purpose .

• Negative control: Use one of the following approaches:

  • Tissue known not to express GRIN1

  • GRIN1 knockout tissue (e.g., from Grin1 ΔBLA mice as described in research)

  • Primary antibody omission control

• Isotype control: Include a non-specific rabbit IgG conjugated to FITC at the same concentration as the primary antibody.

• Absorption control: Pre-incubate the antibody with excess recombinant GRIN1 protein (274-451AA) to block specific binding sites .

• Autofluorescence control: Examine unstained tissue to assess natural autofluorescence that could interfere with FITC signal interpretation.

How can I reduce background fluorescence when using FITC-conjugated antibodies in immunofluorescence?

High background is a common challenge when working with FITC-conjugated antibodies. To minimize this issue:

• Optimize blocking: Use a blocking solution containing 5-10% serum from the same species as the secondary antibody (if using one) or from a different species than the host of the primary antibody.

• Include detergents: Add 0.1-0.3% Triton X-100 or 0.05% Tween-20 to reduce non-specific binding.

• Autofluorescence reduction:

  • For fixed tissues, treat with 0.1-1% sodium borohydride for 5-10 minutes before antibody incubation

  • Use Sudan Black B (0.1-0.3% in 70% ethanol) after antibody staining

  • Consider TrueBlack® or similar commercial autofluorescence quenchers

• Optimize antibody concentration: Titrate the antibody to find the concentration that gives the best signal-to-noise ratio.

• Washing steps: Increase the number and duration of washing steps (using PBS with 0.05-0.1% Tween-20).

• Filter the antibody: Centrifuge at high speed or filter through a 0.22 μm filter before use to remove aggregates.

What alternative antigen retrieval methods can I try if standard protocols don't work with GRIN1 Antibody, FITC conjugated?

If standard antigen retrieval methods aren't yielding satisfactory results, consider these alternative approaches:

• Heat-induced epitope retrieval (HIER) modifications:

  • Try TE buffer at pH 9.0 as suggested for similar GRIN1 antibodies

  • Alternatively, test citrate buffer at pH 6.0

  • Vary the heating time (10-30 minutes) and temperature (90-125°C)

  • Use different heating methods (microwave, pressure cooker, water bath)

• Enzymatic antigen retrieval:

  • Proteinase K (5-20 μg/ml for 5-15 minutes at room temperature)

  • Trypsin (0.05-0.1% for 10-20 minutes at 37°C)

  • Pepsin (0.1-0.5 mg/ml in 0.01N HCl for 5-15 minutes at 37°C)

• Combined approaches:

  • Sequential use of enzymatic digestion followed by HIER

  • Dual buffer system with a transition from acidic to basic conditions

Remember to validate each method with appropriate controls to ensure specificity is maintained.

How can I preserve FITC fluorescence intensity during long experimental protocols?

FITC is susceptible to photobleaching and degradation during extended experimental procedures. To maintain signal intensity:

• Minimize light exposure:

  • Work in reduced ambient lighting

  • Cover samples with aluminum foil during incubations

  • Limit exposure during microscopy by using neutral density filters and fast capture settings

• Add anti-fade agents:

  • Include anti-fade reagents in mounting media (e.g., p-phenylenediamine, n-propyl gallate)

  • Use commercial anti-fade mounting media specifically designed for FITC preservation

• Optimize fixation:

  • Use 2-4% paraformaldehyde (avoid glutaraldehyde which increases autofluorescence)

  • Limit fixation time to minimize epitope masking

• Sample storage:

  • Store slides at 4°C in the dark

  • For long-term storage, consider -20°C after thorough drying of mounting media

• Chemical additives:

  • Add 10 mM sodium azide to buffers (note: toxic, handle with care)

  • Include antioxidants such as 1-4 mg/ml ascorbic acid in buffer solutions

How can GRIN1 Antibody, FITC conjugated be used to investigate NMDAR trafficking and localization in neurons?

GRIN1 Antibody, FITC conjugated offers powerful capabilities for studying the dynamic distribution and trafficking of NMDA receptors in neuronal systems:

• Live cell imaging: The direct FITC conjugation enables real-time visualization of GRIN1 surface expression and internalization in living neurons without permeabilization. This approach can be combined with temperature shifts or drug treatments to track receptor trafficking under various conditions.

• Synaptic vs. extrasynaptic localization: Co-labeling with synaptic markers (such as PSD-95 or synaptophysin) using antibodies conjugated to spectrally distinct fluorophores (e.g., Cy3) allows quantification of the relative distribution of GRIN1-containing NMDARs between synaptic and extrasynaptic compartments .

• Endocytic pathway tracking: Combined with organelle-specific markers, GRIN1 Antibody, FITC conjugated can reveal the intracellular fate of internalized receptors through colocalization analysis.

• Activity-dependent redistribution: By applying chemical LTP/LTD protocols or utilizing optogenetic stimulation, researchers can investigate how synaptic activity regulates GRIN1-containing NMDAR localization and membrane insertion.

• Super-resolution microscopy: Though FITC is not ideal for some super-resolution techniques, it can be used with structured illumination microscopy (SIM) to achieve resolution beyond the diffraction limit, revealing nanoscale organization of NMDAR clusters.

What approaches can be used to validate the specificity of GRIN1 Antibody, FITC conjugated in complex experimental systems?

Validating antibody specificity is crucial for reliable data interpretation. For GRIN1 Antibody, FITC conjugated, consider these advanced validation approaches:

• Genetic knockout/knockdown verification:

  • Use tissue from conditional Grin1 knockout mice (such as Grin1 ΔBLA) as negative controls

  • Compare staining patterns in tissues treated with Grin1-specific siRNA or shRNA

  • Employ CRISPR-Cas9 edited cell lines with GRIN1 deletions

• Orthogonal detection methods:

  • Validate findings using in situ hybridization to detect Grin1 mRNA in parallel with antibody staining

  • Compare results with a different antibody against GRIN1 that recognizes a distinct epitope

  • Correlate protein detection with functional assays of NMDAR activity

• Absorption tests with specific peptide sequences:

  • Pre-incubate the antibody with the immunogen peptide (recombinant Human Glutamate receptor ionotropic, NMDA 1 protein, amino acids 274-451)

  • Compare signal reduction across different concentrations of competing peptide

• Mass spectrometry verification:

  • Perform immunoprecipitation using the antibody followed by mass spectrometry analysis

  • Confirm that GRIN1 is the predominant protein detected

• Cross-reactivity assessment:

  • Test against tissues expressing other NMDAR subunits but not GRIN1

  • Compare staining patterns across multiple species with known sequence differences

How can GRIN1 Antibody, FITC conjugated be integrated into multiplexed imaging approaches?

Multiplexed imaging allows visualization of multiple targets simultaneously, providing contextual information about GRIN1 expression and interaction networks:

• Spectral compatibility considerations:

  • FITC has excitation/emission peaks at approximately 495/519 nm

  • Pair with fluorophores having minimal spectral overlap, such as:

    • Cy3 (550/570 nm) for dual labeling

    • Cy5 (650/670 nm) or AF647 for three-color imaging

    • DAPI (350/470 nm) for nuclear counterstaining

• Sequential labeling approaches:

  • For highly complex multiplex imaging, consider cyclic immunofluorescence with antibody stripping

  • Photobleach FITC after imaging, then reprobe with additional antibodies

• Combination with RNA detection:

  • Integrate with fluorescence in situ hybridization (FISH) using FITC-UTP or DIG-UTP labeled cRNA probes

  • Perform dual protein-RNA detection to correlate GRIN1 protein localization with mRNA expression

• Tissue clearing techniques:

  • Combine with CLARITY, iDISCO, or other clearing methods for 3D volumetric imaging

  • Optimize fixation and clearing protocols to preserve FITC fluorescence

• Multimodal imaging integration:

  • Correlate fluorescence microscopy with electron microscopy using immunogold labeling

  • Combine with functional calcium imaging to link GRIN1 distribution to neuronal activity

What are the comparative advantages and limitations of using FITC-conjugated versus unconjugated GRIN1 antibodies?

Understanding the tradeoffs between directly conjugated and unconjugated antibodies helps in selecting the optimal approach for specific research questions:

For GRIN1 detection specifically, unconjugated antibodies have demonstrated reliable performance in Western Blot (1:2000-1:10000 dilution), IHC (1:500-1:2000), and IF-P (1:250-1:1000) .

How does FITC compare with other fluorophores for GRIN1 detection in neuroscience applications?

Different fluorophores offer varying properties that may be advantageous depending on the specific experimental context:

For neuroscience applications specifically, consider:

• Use Alexa Fluor 488 instead of FITC when performing lengthy confocal imaging sessions

• Choose far-red fluorophores when working with brain tissue, which has significant autofluorescence in the green-yellow spectrum

• Consider photoconvertible fluorophores for pulse-chase experiments tracking NMDAR trafficking

How do different tissue preparation techniques affect the detection of GRIN1 using FITC-conjugated antibodies?

The choice of tissue preparation methodology significantly impacts the quality and reliability of GRIN1 detection:

For brain tissue specifically, research has shown that:

• Antigen retrieval with TE buffer at pH 9.0 is often effective for GRIN1 detection

• Citrate buffer at pH 6.0 can be used as an alternative retrieval method

• Expression patterns can be verified using dual detection with in situ hybridization for Grin1 mRNA

How should researchers quantify and analyze GRIN1 expression patterns in immunofluorescence studies?

Quantitative analysis of GRIN1 immunolabeling requires careful consideration of image acquisition and analysis parameters:

What are the known pitfalls in interpreting GRIN1 antibody signals in different experimental contexts?

Several factors can complicate the interpretation of GRIN1 immunolabeling:

• Splice variant considerations:

  • GRIN1 has multiple splice variants with different tissue distributions

  • Verify which epitope region (274-451AA) is recognized by your antibody and whether it covers all relevant isoforms

• Post-translational modifications:

  • Phosphorylation, glycosylation, or other modifications may mask epitopes

  • Consider using phospho-specific antibodies if studying activity-dependent regulation

• Receptor internalization effects:

  • Surface vs. intracellular pools may require different detection protocols

  • Use non-permeabilized conditions to specifically detect surface expression

• Cross-reactivity concerns:

  • Verify specificity against other glutamate receptor family members

  • Use knockout controls or absorption tests to confirm signal specificity

• Signal interpretation challenges:

  • FITC signal can be confused with lipofuscin autofluorescence in aged brain tissue

  • Low signal-to-noise ratio may require signal amplification methods

  • Punctate vs. diffuse staining patterns reflect different biological states

• Technical artifacts:

  • Edge effects and tissue folding can create false positive signals

  • Insufficient blocking can lead to non-specific binding

  • Antibody aggregates may appear as punctate signals

How can GRIN1 Antibody, FITC conjugated be used to investigate NMDAR dysfunction in neurological disorders?

GRIN1 Antibody, FITC conjugated provides valuable tools for studying NMDAR abnormalities in disease states:

• Altered expression patterns:

  • Quantify changes in GRIN1 levels in post-mortem tissue from patients with neurological disorders

  • Compare cellular and subcellular distribution between control and disease tissues

  • Correlate expression changes with disease progression markers

• Subunit composition analysis:

  • Co-label with antibodies against other NMDAR subunits to assess changes in receptor composition

  • Evaluate the ratio of GRIN1 to other subunits (GluN2A, GluN2B) which affects receptor function

  • Analyze developmental switches in subunit expression in neurodevelopmental disorders

• Experimental disease models:

  • Use in transgenic mouse models (e.g., Grin1 ΔBLA) to study the effects of NMDAR dysfunction

  • Apply to in vitro models of neurodegeneration, excitotoxicity, or hypoxia

  • Monitor dynamic changes in receptor trafficking during disease progression

• Intervention assessment:

  • Evaluate how pharmacological agents affect GRIN1 expression and localization

  • Monitor receptor recovery following therapeutic interventions

  • Track changes in NMDAR function in response to neuroprotective strategies

• Circuit-specific analysis:

  • Investigate region-specific alterations in GRIN1 distribution

  • Analyze cell-type specific changes using co-labeling with neuronal subtype markers

  • Assess synaptic vs. extrasynaptic receptor balance, which is often disrupted in disease states