GPHN Antibody, FITC conjugated

What is Gephyrin (GPHN) and what are its biological functions?

Gephyrin (GPHN) is a multifunctional microtubule-associated protein that plays critical roles in both synapse organization and enzymatic pathways. It functions primarily as a scaffolding protein involved in membrane protein-cytoskeleton interactions, most notably anchoring inhibitory glycine receptors (GLYR) to subsynaptic microtubules. This anchoring function is essential for maintaining proper inhibitory neurotransmission in the central nervous system. Beyond its structural role, GPHN also functions as a bifunctional enzyme that catalyzes two crucial steps in the biosynthesis of the molybdenum cofactor. Initially, it adenylates molybdopterin (functioning as molybdopterin adenylyltransferase, EC 2.7.7.75) through its G domain, followed by insertion of molybdate into the adenylated molybdopterin with concurrent AMP release (functioning as molybdopterin molybdenumtransferase, EC 2.10.1.1) via its E domain .

What are the key specifications of commercially available GPHN Antibody, FITC conjugated?

Commercial GPHN Antibody, FITC conjugated is typically supplied as a research-grade reagent with the following specifications:

• Source organism: Rabbit

• Isotype: IgG

• Target reactivity: Human

• Conjugate: Fluorescein isothiocyanate (FITC)

• Standard quantity: 50μg

• Immunogen: Recombinant Human Gephyrin protein (typically amino acids 360-460)

• Storage buffer composition: 50% Glycerol, 0.01M PBS (pH 7.4), with 0.03% Proclin 300 as preservative

• Primary validated application: ELISA

• Protein identifier: UniProt Q9NQX3

How does FITC conjugation affect antibody functionality and what mechanisms underlie these effects?

FITC conjugation involves the covalent attachment of fluorescein isothiocyanate molecules to reactive lysine residues on antibody proteins. This labeling process can potentially alter antibody functionality through several mechanisms. The rate and extent of labeling varies with reaction conditions, label concentration, and the nature of the target protein. While FITC provides the necessary fluorescent properties for visualization, excessive labeling can impact antibody binding characteristics.

What validated research applications are appropriate for GPHN Antibody, FITC conjugated?

GPHN Antibody, FITC conjugated has been validated for multiple research applications in neuroscience, cancer research, and basic cell biology. The primary validated applications include:

• Enzyme-Linked Immunosorbent Assay (ELISA): The antibody has been specifically validated for quantitative detection of human GPHN in various sample types .

• Immunohistochemistry (IHC): Extensive validation demonstrates utility in paraffin-embedded sections of diverse tissue types including:

  • Human pathological samples (liver cancer, papillary thyroid carcinoma, stomach cancer)

  • Normal rodent tissues (mouse and rat brain, rat lung)

• Immunofluorescence (IF): The antibody has been successfully employed in immunocytochemical analysis of cultured cell lines such as A431 cells, where FITC labeling provides direct visualization without secondary antibody requirements .

• Novel analytical applications: The FITC conjugation enables specialized applications such as antibody-antigen binding analysis via modified differential scanning fluorimetry (DSF) without requiring additional extrinsic fluorophores .

What is the optimal protocol for immunohistochemistry using GPHN Antibody, FITC conjugated?

For optimal immunohistochemical detection of GPHN using FITC-conjugated antibody, the following methodological approach is recommended:

• Tissue preparation and antigen retrieval:

  • Use paraffin-embedded sections (4-6 μm thickness)

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

  • Ensure complete deparaffinization and rehydration prior to retrieval

• Blocking and primary antibody incubation:

  • Block tissue sections with 10% goat serum for 30-60 minutes at room temperature

  • Incubate with GPHN Antibody, FITC conjugated at 2 μg/ml concentration

  • Maintain incubation overnight (12-16 hours) at 4°C in a humidified chamber

  • For FITC direct detection, proceed to counterstaining and mounting

• Alternative detection method for amplified signal:

  • If signal amplification is required, use biotinylated goat anti-rabbit IgG as secondary antibody

  • Incubate for precisely 30 minutes at 37°C

  • Develop using Strepavidin-Biotin-Complex with DAB as the chromogen

  • Note: This approach sacrifices the direct fluorescence of FITC but provides signal amplification

How can differential scanning fluorimetry be optimized for antigen binding studies with FITC-labeled antibodies?

A novel approach to differential scanning fluorimetry (DSF) leverages the intrinsic fluorescence of FITC-conjugated antibodies without requiring additional extrinsic fluorophores. This method has significant advantages for studying antigen binding when standard DSF approaches would face spectral interference. The optimized protocol includes:

• Sample preparation:

  • Prepare FITC-conjugated antibody (15-120 minute labeling time is optimal)

  • Create concentration series of target antigen

  • Combine antibody with varying antigen concentrations in appropriate buffer

• Instrument settings:

  • Configure real-time PCR thermocycler with appropriate excitation/emission filters for FITC

  • Set temperature ramp rate to 1°C/minute from 25°C to 95°C

  • Program fluorescence data collection at each temperature increment

• Data analysis approach:

  • Unlike traditional DSF where fluorescence increases upon protein unfolding, FITC fluorescence decreases during thermal denaturation

  • Plot fluorescence versus temperature for each antigen concentration

  • Determine thermal stability shifts (ΔTm) as a function of antigen concentration

  • These shifts quantitatively reflect binding affinity when properly analyzed

This methodology provides a valuable alternative when spectral overlap precludes the use of standard DSF dyes like Sypro orange or DASPMI. The concentration-dependent increase in thermal stability correlates with antigen binding strength, enabling quantitative binding analysis without interference from the FITC label .

How does the degree of FITC labeling impact antibody performance, and what is the optimal labeling ratio?

FITC labeling exhibits time-dependent incorporation patterns that significantly impact antibody performance. Research examining the relationship between labeling duration and antibody function reveals several critical considerations:

The distribution of FITC labeling varies between antibody domains, with preferential early incorporation into Fab fragments followed by increasing labeling of Fc regions over extended reaction times. Optimal performance typically occurs with moderate labeling (15-minute reaction time) that provides sufficient fluorescence while minimizing functional impact. Quantitative analysis using isothermal titration calorimetry demonstrates that even heavily labeled antibodies (120-minute reaction time) retain approximately 90-95% of their binding capacity.

The thermodynamic binding parameters (ΔH, ΔS, ΔG) show minimal alterations with moderate labeling but may shift more significantly with extensive labeling. This reflects subtle conformational changes that can influence binding kinetics. For most research applications, a moderate FITC:antibody ratio that balances detection sensitivity and binding preservation is recommended .

What are the most effective strategies for reducing background fluorescence when using GPHN Antibody, FITC conjugated?

Background fluorescence can significantly impact signal-to-noise ratios when working with FITC-conjugated antibodies. Implementing these methodological refinements can substantially improve specificity:

• Optimized blocking protocol:

  • Extend blocking time to 60 minutes with freshly prepared 10% serum from the same species as the secondary antibody (if used)

  • Include 0.1-0.3% Triton X-100 in blocking solution to reduce non-specific membrane binding

  • Consider adding 1% BSA to further reduce background interactions

• Antibody dilution optimization:

  • Perform systematic titration experiments to determine minimum effective concentration

  • For most applications with GPHN Antibody, FITC conjugated, 2 μg/ml represents an optimal starting point

  • Prepare antibody dilutions in blocking buffer rather than plain PBS

• Specific countermeasures for tissue autofluorescence:

  • Pretreat tissues with 0.1-1% sodium borohydride solution to quench aldehyde-induced autofluorescence

  • Include brief Sudan Black B treatment (0.1-0.3% in 70% ethanol) after antibody incubation

  • Consider photobleaching samples briefly before antibody addition to reduce endogenous fluorescence

How can the specificity of GPHN Antibody, FITC conjugated be validated in experimental systems?

Rigorous validation of GPHN Antibody, FITC conjugated specificity requires a multi-faceted approach:

• Tissue distribution pattern analysis:

  • Compare antibody localization with established GPHN expression patterns

  • GPHN shows characteristic punctate distribution at inhibitory synapses in neuronal tissues

  • Verify appropriate subcellular localization (primarily postsynaptic in neurons)

• Technical validation controls:

  • Include absorption controls using recombinant GPHN protein (specifically amino acids 360-460)

  • Perform parallel staining with alternative validated anti-GPHN antibodies

  • Include tissues/cells known to be negative for GPHN expression

• Advanced validation approaches:

  • Compare staining in wild-type versus GPHN-knockout models (if available)

  • Employ RNA interference to knockdown GPHN expression and confirm reduced signal

  • Utilize recombinant GPHN expression systems to verify detection sensitivity

• Quantitative assessment:

  • Perform Western blot analysis in parallel with fluorescence detection

  • Verify detection of the expected ~80 kDa band corresponding to full-length GPHN

  • Assess cross-reactivity with potential splice variants known to exist for GPHN

How can isothermal titration calorimetry be used to quantify the effects of FITC conjugation on antibody-antigen interactions?

Isothermal titration calorimetry (ITC) offers a powerful, label-independent approach to quantitatively assess how FITC conjugation affects antibody-antigen binding thermodynamics. Unlike fluorescence-based methods, ITC is not subject to interference from the FITC label, providing direct measurement of fundamental binding parameters. The methodology involves:

• Experimental design for comparative analysis:

  • Analyze unlabeled control antibody, moderately labeled (15 min), and extensively labeled (120 min) samples under identical conditions

  • Titrate identical antigen preparations into each antibody sample

  • Maintain consistent buffer conditions, temperature, and instrumentation parameters

• Key parameters for quantitative assessment:

  • Binding stoichiometry (N): Reveals potential reduction in functional binding sites

  • Binding affinity (Ka/Kd): Quantifies changes in interaction strength

  • Enthalpy change (ΔH): Measures alterations in hydrogen bonding and van der Waals interactions

  • Entropy change (ΔS): Reflects changes in hydrophobic interactions and conformational flexibility

• Data interpretation framework:

  • Minor changes in N values (5-10% reduction) indicate minimal steric hindrance from FITC

  • Shifts in thermodynamic profile (entropy-enthalpy compensation) suggest subtle conformational changes

  • Preservation of Gibbs free energy (ΔG) despite changes in ΔH and ΔS indicates compensatory mechanisms maintaining binding

Published research demonstrates that moderate FITC labeling typically preserves most binding capacity, with only subtle alterations in thermodynamic parameters, making properly labeled antibodies suitable for most research applications .

What novel analytical approaches can be combined with GPHN Antibody, FITC conjugated for studying inhibitory synapse dynamics?

Advanced research into inhibitory synapse dynamics can leverage GPHN Antibody, FITC conjugated through several innovative methodological combinations:

• Live-cell imaging with complementary markers:

  • Combine GPHN Antibody, FITC conjugated with membrane-permeable voltage-sensitive dyes

  • Pair with spectrally distinct fluorophore-conjugated antibodies against inhibitory neurotransmitter receptors (GABA-A, glycine receptors)

  • Integrate with genetically encoded calcium indicators to correlate structural and functional data

• Super-resolution microscopy applications:

  • Implement Stimulated Emission Depletion (STED) microscopy to resolve nanoscale distribution patterns of gephyrin scaffolds

  • Apply Stochastic Optical Reconstruction Microscopy (STORM) to quantify molecular clustering and stoichiometry

  • Combine with expansion microscopy to enhance spatial resolution of inhibitory synapse architecture

• Correlative microscopy approaches:

  • Utilize GPHN Antibody, FITC conjugated for initial fluorescence identification of regions of interest

  • Subsequently process the same samples for electron microscopy to analyze ultrastructural details

  • Implement fiducial markers to precisely align fluorescence and electron microscopy datasets

• Functional analysis integration:

  • Apply optical uncaging of neurotransmitters near identified gephyrin clusters

  • Combine with electrophysiological recording to correlate structural and functional properties

  • Implement optogenetic manipulation of inhibitory circuits while monitoring gephyrin dynamics

How can structural analysis techniques determine the impact of FITC labeling on GPHN antibody binding epitopes?

Understanding the structural implications of FITC conjugation on antibody-antigen interactions requires sophisticated analytical approaches:

• High-resolution epitope mapping:

  • Employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions where FITC labeling alters protein dynamics

  • Utilize peptide microarrays with overlapping GPHN peptide fragments to precisely map binding epitopes

  • Compare epitope accessibility in labeled versus unlabeled antibody preparations

• Computational modeling approaches:

  • Generate molecular models of GPHN-antibody complexes

  • Simulate the effects of lysine modification at different positions

  • Calculate binding energy changes resulting from FITC conjugation

• Crystallographic and cryo-EM analysis:

  • Obtain high-resolution structures of Fab fragments with and without FITC labeling

  • Compare electron density maps to identify conformational changes

  • Assess alterations in complementarity-determining region (CDR) orientation

• Site-directed mutagenesis validation:

  • Systematically mutate lysine residues near the binding interface to non-reactive residues

  • Evaluate the impact of these mutations on binding compared to FITC labeling

  • Confirm computational predictions through experimental binding studies

What are the advantages and limitations of FITC-conjugated GPHN antibody compared to other fluorophore conjugates?

When designing experiments using fluorophore-conjugated GPHN antibodies, researchers should consider these comparative factors:

FITC conjugation offers excellent cost-effectiveness and well-established protocols but presents challenges in applications requiring extended imaging, low pH environments, or complex multiplexing. For standard immunohistochemistry and basic fluorescence microscopy, FITC remains highly suitable, while advanced imaging applications may benefit from alternative fluorophores with enhanced stability and spectral properties .

What methodological modifications are necessary when using GPHN Antibody, FITC conjugated across different experimental systems?

Optimizing experimental protocols for GPHN Antibody, FITC conjugated across diverse biological systems requires systematic adaptations:

• Tissue-specific considerations:

  • Brain tissue: Extend fixation time to 24-48 hours; implement antigen retrieval in EDTA buffer (pH 8.0)

  • Cancer tissues: Modify blocking to include 0.3% hydrogen peroxide to quench endogenous peroxidase

  • Cell cultures: Reduce fixation to 10-15 minutes with 4% PFA to preserve epitope accessibility

• Species-specific optimizations:

  • Human samples: Standard protocol (2 μg/ml antibody) typically sufficient

  • Rodent tissues: May require increased antibody concentration (3-4 μg/ml) for equivalent signal

  • Non-mammalian models: Validate cross-reactivity and optimize fixation conditions empirically

• Application-dependent modifications:

  • For IHC: Consider signal amplification via biotin-streptavidin systems when needed

  • For live imaging: Utilize Fab fragments of FITC-conjugated antibody to improve tissue penetration

  • For super-resolution: Implement additional post-fixation steps to minimize sample movement

• Counterstain compatibility considerations:

  • With DAPI: Standard protocol suitable

  • With propidium iodide: Implement RNase treatment to reduce background

  • With multiple antibodies: Carefully sequence application to minimize cross-reactivity

How can contradictory experimental results using GPHN Antibody, FITC conjugated be systematically analyzed and resolved?

When confronted with inconsistent or contradictory results using GPHN Antibody, FITC conjugated, implement this systematic troubleshooting framework:

• Authentication and validation phase:

  • Verify antibody specificity via Western blot against recombinant GPHN

  • Confirm fluorophore activity through spectroscopic analysis

  • Assess lot-to-lot variation through comparative testing

• Technical variables assessment:

  • Systematically evaluate fixation parameters (duration, temperature, fixative composition)

  • Test multiple antigen retrieval methods (heat-mediated vs. enzymatic; buffer composition)

  • Document microscopy parameters (exposure time, gain settings, filter configurations)

• Biological variability analysis:

  • Consider developmental stage differences in GPHN expression patterns

  • Evaluate pharmacological treatments that might alter GPHN clustering

  • Assess regional variations in expression across brain structures

• Methodological cross-validation:

  • Compare results with alternative anti-GPHN antibodies

  • Implement parallel RNA analysis (in situ hybridization or RT-PCR)

  • Utilize genetically labeled GPHN models when available

• Statistical approach to reconciliation:

  • Increase biological and technical replicates

  • Implement blinded quantification protocols

  • Apply appropriate statistical tests to determine significance of observed differences