MAGI2 Antibody, Biotin conjugated

What is MAGI2 and why is it significant in neuroscience research?

MAGI2 (Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2) is a scaffolding protein also known as Atrophin-1-interacting protein 1 (AIP-1), Atrophin-1-interacting protein A, and Membrane-associated guanylate kinase inverted 2 (MAGI-2) . The protein plays crucial roles in neuronal signaling and synaptic organization, making it a significant target for neuroscience investigations. MAGI2 contains multiple protein-protein interaction domains, allowing it to function as a molecular scaffold that facilitates the assembly of signaling complexes at neuronal synapses. Its interactions with various neuronal proteins contribute to synaptic plasticity and neurotransmission, processes fundamental to learning, memory, and other cognitive functions. Studying MAGI2 using specific antibodies enables researchers to investigate its localization, expression patterns, protein interactions, and functional implications in normal neural development and neurological disorders.

What are the key characteristics of commercially available MAGI2 Antibody, Biotin conjugated?

The MAGI2 Antibody, Biotin conjugated is a high-quality polyclonal antibody with specific reactivity against human MAGI2 samples . It is generated in rabbits using a recombinant Human MAGI2 protein fragment (amino acids 1308-1455) as the immunogen . Key specifications include:

• Host Species: Rabbit

• Clonality: Polyclonal

• Isotype: IgG

• Conjugate: Biotin

• Species Reactivity: Human

• Purification Method: Protein G purification (>95% purity)

• Form: Liquid

• Buffer Composition: 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4

• UniProt ID: Q86UL8

• Validated Applications: ELISA

The biotin conjugation provides significant advantages for detection systems, allowing for amplification of signals through the strong biotin-streptavidin interaction, enhancing experimental sensitivity without affecting the antibody's binding properties.

What are the recommended storage and handling conditions for maintaining MAGI2 Antibody, Biotin conjugated integrity?

Proper storage and handling of MAGI2 Antibody, Biotin conjugated is critical for maintaining its functionality and extending its usable lifespan. Upon receipt, the antibody should be stored at either -20°C or -80°C . Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation, aggregation, and loss of antibody functionality .

For routine laboratory use, consider the following handling recommendations:

• Aliquot the antibody into smaller volumes based on experimental needs to minimize freeze-thaw cycles

• When thawing, allow the antibody to come to room temperature slowly

• Brief centrifugation after thawing is recommended to collect all liquid at the bottom of the tube

• Handle the antibody on ice when preparing dilutions

• For short-term use (within 1-2 weeks), the antibody can be stored at 4°C

• Protect biotin-conjugated antibodies from prolonged exposure to light to prevent photobleaching

These storage and handling practices help preserve the integrity of the antibody-biotin conjugate, ensuring consistent experimental results throughout the antibody's shelf life.

What are the methodological advantages of biotin-conjugated MAGI2 antibody compared to unconjugated alternatives?

Biotin conjugation of MAGI2 antibody offers several methodological advantages for neuroscience researchers seeking enhanced detection sensitivity and experimental flexibility:

• Signal Amplification: The biotin-streptavidin system provides one of the strongest non-covalent biological interactions (K​d ≈ 10^-15 M), enabling significant signal amplification in detection systems . This is particularly valuable when studying proteins like MAGI2 that may be expressed at relatively low levels in certain neural tissues.

• Compatibility with Multiple Detection Systems: Biotin-conjugated antibodies can be detected using various streptavidin-conjugated reporter molecules (HRP, fluorophores, gold particles), allowing researchers to adapt their detection strategy to different experimental platforms without changing the primary antibody .

• Reduced Background in Immunohistochemistry: In neural tissue sections with high endogenous peroxidase activity, biotin-conjugated antibodies used with streptavidin-coupled fluorophores can circumvent the background issues associated with HRP-based detection.

• Multiplexing Capability: When combined with other directly-labeled primary antibodies, biotin-conjugated MAGI2 antibody facilitates multi-protein co-localization studies, which are essential for understanding MAGI2's scaffolding functions in complex neuronal signaling networks.

• Enhanced Sensitivity in ELISA: The validated ELISA application of this antibody can be optimized through biotin-streptavidin amplification systems, lowering detection thresholds for MAGI2 in biological samples.

When compared to unconjugated alternatives, the biotin-conjugated format eliminates the need for species-specific secondary antibodies, reducing potential cross-reactivity issues in multi-species experimental systems common in neuroscience research.

How should researchers optimize ELISA protocols when using MAGI2 Antibody, Biotin conjugated?

Optimizing ELISA protocols with MAGI2 Antibody, Biotin conjugated requires systematic consideration of multiple parameters to achieve maximum sensitivity and specificity:

Table 1: ELISA Optimization Parameters for MAGI2 Antibody, Biotin Conjugated

Methodological Considerations:

• Plate Preparation: Coat high-binding ELISA plates with either recombinant MAGI2 (for antibody validation) or capture antibody (for sandwich ELISA detection of native MAGI2).

• Blocking: Given the composition of the antibody buffer (containing 50% glycerol), thorough blocking is essential to minimize background. A 3% BSA solution in PBS is recommended as an initial blocking condition .

• Detection Amplification: For enhanced sensitivity, consider using poly-HRP-streptavidin conjugates which can significantly lower detection thresholds.

• Controls: Always include:

  • A standard curve using recombinant MAGI2 protein

  • A negative control using an isotype-matched biotin-conjugated antibody

  • A control omitting the primary antibody to assess detection system background

• Data Analysis: Calculate the limit of detection (LoD) and limit of quantification (LoQ) based on the standard deviation of blank samples to ensure reliable interpretation of experimental results.

By systematically optimizing these parameters, researchers can develop robust ELISA protocols for MAGI2 detection suitable for their specific experimental requirements.

What strategies can be employed to validate the specificity of MAGI2 Antibody, Biotin conjugated in experimental systems?

Validating the specificity of MAGI2 Antibody, Biotin conjugated is crucial for ensuring reliable experimental results, particularly in neuroscience applications where cross-reactivity could lead to misinterpretation. A comprehensive validation strategy should include:

• Western Blot Analysis: Though not explicitly listed in the validated applications, Western blotting with human neural cell/tissue lysates should detect a band corresponding to MAGI2's molecular weight (~140-150 kDa). Comparison with lysates from cells where MAGI2 has been knocked down via siRNA provides a critical negative control.

• Immunoprecipitation Followed by Mass Spectrometry: Perform immunoprecipitation with the MAGI2 antibody and analyze the captured proteins by mass spectrometry to confirm pull-down of MAGI2 and identify any potential cross-reactive proteins.

• Immunocytochemistry with Overexpression Systems: Transfect cells with MAGI2-expressing constructs (tagged with a different reporter like GFP) and demonstrate co-localization of antibody signal with the overexpressed protein.

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide (amino acids 1308-1455 of human MAGI2) before application in your experimental system; specific binding should be significantly reduced or eliminated.

• Cross-Species Reactivity Testing: Although the antibody is specified for human reactivity, testing with conserved regions from other species can help understand potential broader applications and confirm specificity.

• Comparison with Alternative MAGI2 Antibodies: Compare detection patterns with other validated MAGI2 antibodies targeting different epitopes to corroborate findings.

• Correlation with mRNA Expression: In tissue panels or developmental series, antibody detection patterns should correlate with mRNA expression profiles of MAGI2 as determined by RT-PCR or RNA-seq data.

These validation approaches collectively provide strong evidence for antibody specificity and help establish appropriate experimental conditions and controls for subsequent studies focusing on MAGI2 biology in neuronal systems.

How does site-specific conjugation technology compare to conventional methods for antibody functionalization?

The conventional methods for antibody conjugation typically involve random targeting of amino groups on lysine residues or thiol groups on cysteine residues, resulting in heterogeneous products with undefined stoichiometry and considerable batch-to-batch variability . In contrast, site-specific conjugation technologies, such as selenocysteine interface technology, offer significant advantages for creating precisely defined antibody conjugates.

Table 2: Comparison of Antibody Conjugation Methods

Selenocysteine interface technology offers particularly notable advantages:

• It involves only minor modifications at the C-terminus that do not interfere with disulfide bridges crucial for antibody structure

• It does not require chemical activation steps that might compromise antibody function

• It generates unique 1:1 stoichiometries of biological and chemical components

For researchers working with specialized applications of MAGI2 antibodies, site-specific conjugation could offer improved reproducibility and performance in applications requiring precise quantification or where random conjugation might compromise epitope recognition. While conventional biotin conjugation is suitable for many standard applications, researchers pursuing advanced applications like super-resolution imaging, development of antibody-drug conjugates, or quantitative proteomics might benefit from the enhanced precision of site-specific conjugation methods.

What are common pitfalls when using MAGI2 Antibody, Biotin conjugated in immunohistochemistry and how can they be addressed?

Although ELISA is the validated application for the MAGI2 Antibody, Biotin conjugated, researchers may adapt it for immunohistochemistry (IHC) studies. Several challenges specific to this application with biotin-conjugated antibodies require particular attention:

• Endogenous Biotin Interference: Neural tissues often contain high levels of endogenous biotin, which can cause significant background when using biotin-streptavidin detection systems.

  • Solution: Implement an endogenous biotin blocking step using avidin/biotin blocking kits before applying the primary antibody.

• Fixation-Induced Epitope Masking: The MAGI2 epitope (amino acids 1308-1455) may be sensitive to certain fixation protocols, particularly with cross-linking fixatives like formaldehyde.

  • Solution: Optimize fixation conditions (duration, concentration) and evaluate the need for antigen retrieval methods (heat-induced or enzymatic).

• Autofluorescence in Neural Tissues: When using fluorophore-coupled streptavidin, lipofuscin autofluorescence in neural tissues can interfere with specific signal detection.

  • Solution: Treat sections with Sudan Black B (0.1-0.3%) after immunolabeling or use spectral unmixing during confocal microscopy.

• Signal Amplification Artifacts: Excessive amplification through the biotin-streptavidin system can sometimes create artifactual staining patterns.

  • Solution: Titrate both primary antibody and streptavidin-conjugate concentrations to optimize signal-to-noise ratios.

• Cross-Reactivity with Biotin-Containing Proteins: Some biotin-binding proteins in tissues may non-specifically interact with the biotin on the antibody.

  • Solution: Include additional blocking steps with irrelevant biotinylated proteins.

• Cell Permeabilization Challenges: MAGI2 localization at membrane junctions may require specific permeabilization approaches for optimal antibody access.

  • Solution: Compare detergent-based (Triton X-100, saponin) versus solvent-based (methanol) permeabilization methods.

A systematic approach to addressing these challenges, with appropriate controls at each step, will help establish reliable IHC protocols for visualizing MAGI2 in neural tissues despite these technical considerations.

How can researchers integrate MAGI2 Antibody, Biotin conjugated into multi-parameter analyses for neuroscience research?

Integrating MAGI2 Antibody, Biotin conjugated into multi-parameter analyses enables comprehensive investigation of MAGI2's role within complex neuronal networks and signaling pathways. Several advanced approaches can be considered:

• Multiplexed Immunofluorescence:

  • Combine MAGI2 detection with other neuronal markers using streptavidin-coupled fluorophores with spectral properties distinct from directly-labeled antibodies against synaptic proteins (e.g., PSD-95, Synapsin).

  • Implement sequential staining protocols using tyramide signal amplification (TSA) for multi-epitope detection with antibodies from the same species.

• Proximity Ligation Assay (PLA):

  • Use the biotin-conjugated MAGI2 antibody with streptavidin-oligonucleotides for PLA to detect and quantify protein-protein interactions between MAGI2 and suspected binding partners in situ.

  • This approach provides spatial resolution of interactions at specific subcellular compartments in neurons.

• Flow Cytometry for Neural Cell Subtyping:

  • Adapt the MAGI2 antibody for flow cytometry by using streptavidin-fluorophores compatible with available cytometer channels.

  • Combine with neural stem cell or neuronal subtype markers to study MAGI2 expression across different neural populations.

• Mass Cytometry (CyTOF):

  • Utilize metal-conjugated streptavidin for detection of the biotinylated MAGI2 antibody in CyTOF experiments.

  • This approach allows simultaneous detection of 30+ parameters for comprehensive neural phenotyping.

• Spatial Transcriptomics Integration:

  • Correlate MAGI2 protein localization (detected via the antibody) with spatial transcriptomic data to understand regional variations in MAGI2 expression and potential co-regulatory networks.

• Super-Resolution Microscopy:

  • Employ streptavidin conjugated to photo-switchable fluorophores for STORM or PALM super-resolution microscopy.

  • This enables nanoscale visualization of MAGI2 organization at neuronal synapses.

• Functional Correlation:

  • Combine MAGI2 immunodetection with calcium imaging or electrophysiology to correlate protein localization with neuronal activity patterns.

Table 3: Multi-Parameter Analysis Technologies with MAGI2 Antibody

These integrative approaches significantly extend the utility of the MAGI2 Antibody, Biotin conjugated beyond its validated ELISA application, enabling researchers to address complex questions about MAGI2 biology in the nervous system.

What emerging technologies might enhance the utility of MAGI2 Antibody, Biotin conjugated in neuroscience research?

Several emerging technologies hold promise for expanding the applications and enhancing the utility of MAGI2 Antibody, Biotin conjugated in neuroscience research:

• Expansion Microscopy: Physical expansion of biological specimens can improve the resolution of conventional microscopes. The biotin-streptavidin interaction's strength makes biotinylated antibodies particularly suitable for this approach, as they remain bound during the expansion process, enabling improved visualization of MAGI2's subcellular distribution at synaptic structures.

• Microfluidic Antibody Capture: Novel microfluidic platforms for protein analysis could leverage the biotin-conjugated antibody for automated, high-throughput assessment of MAGI2 levels in cerebrospinal fluid or brain microdialysates from experimental models or clinical samples.

• Antibody-Based Biosensors: Integration of the MAGI2 antibody into electrochemical or optical biosensors via biotin-streptavidin coupling could enable real-time monitoring of MAGI2 expression changes in neural cell cultures or organoids.

• CRISPR-Based Tagging Combined with Antibody Detection: Endogenous tagging of MAGI2 using CRISPR-Cas9 technology, followed by detection with the biotin-conjugated antibody, could provide unique insights into the dynamics of native MAGI2 without overexpression artifacts.

• Advanced Tissue Clearing Techniques: Compatibility of the biotin-conjugated antibody with CLARITY, iDISCO, or other tissue clearing methods would enable whole-brain mapping of MAGI2 distribution in developmental stages or disease models.

• In Vivo Neuroimaging: Development of brain-penetrant forms of the antibody or antibody fragments could potentially enable in vivo imaging of MAGI2 in animal models using PET or SPECT when coupled with appropriate isotope-labeled streptavidin.

The evolution of site-specific conjugation technologies like selenocysteine interface technology suggests that future iterations of MAGI2 antibodies might achieve even greater precision in conjugation and functionality, further enhancing their utility in both basic neuroscience research and potential diagnostic applications.