CLIP2 Antibody, FITC conjugated

What is CLIP2 protein and what biological functions does it serve?

CLIP2 (CAP-Gly domain-containing linker protein 2) functions as a critical cytoskeletal linker protein in neuronal cells. It specifically links microtubules to dendritic lamellar bodies (DLBs), which are membranous organelles predominantly found in bulbous dendritic appendages of neurons connected by dendrodendritic gap junctions . This protein appears to operate in the control of brain-specific organelle translocations, suggesting its importance in neuronal function and compartmentalization . CLIP2 is also known by several other names including Cytoplasmic linker protein 115 (CLIP-115), Williams-Beuren syndrome chromosomal region 3 protein, and Williams-Beuren syndrome chromosomal region 4 protein . The protein is encoded by the CLIP2 gene (also known as CYLN2, KIAA0291, WBSCR3, WBSCR4, WSCR4) and has the UniProt identifier Q9UDT6 .

What is the reactivity spectrum and source characteristics of this antibody?

The CLIP2 Antibody, FITC conjugated demonstrates human reactivity, meaning it specifically recognizes human CLIP2 protein . The antibody is derived from rabbit (source) and belongs to the IgG isotype, which provides good stability and consistent performance in most immunological applications . The immunogen used for generating this antibody is a recombinant Human CAP-Gly domain-containing linker protein 2 protein fragment spanning amino acids 101-217 . This specific epitope information is crucial for researchers planning experiments involving protein domains or truncated variants of CLIP2.

How should researchers implement CLIP2 Antibody, FITC conjugated in ELISA protocols?

For optimal ELISA implementation using CLIP2 Antibody, FITC conjugated, researchers should follow these methodological steps:

• Sample Preparation: Prepare protein lysates from human tissue or cell lines known to express CLIP2 (neuronal cells recommended)

• Coating: Coat ELISA plates with capture antibody against CLIP2 or with the sample directly in a sandwich or direct ELISA format

• Blocking: Block non-specific binding sites with appropriate blocking buffer (typically 3-5% BSA in PBS)

• Antibody Application: Apply the FITC-conjugated CLIP2 antibody at the appropriate dilution (determine optimal concentration through titration)

• Detection: Unlike conventional ELISA requiring enzyme-conjugated secondary antibodies, FITC detection requires a fluorescence plate reader with appropriate excitation (492 nm) and emission (520 nm) settings

• Controls: Include positive controls (samples known to contain CLIP2), negative controls (samples lacking CLIP2), and background controls (no primary antibody)

When analyzing results, create a standard curve using recombinant CLIP2 protein of known concentrations to quantify CLIP2 levels in experimental samples accurately.

What site-specific conjugation methods can improve CLIP2 antibody labeling?

Recent advances in antibody conjugation techniques offer significant improvements over traditional random conjugation methods. A methodologically superior approach involves a two-step site-specific enzymatic reaction:

• Deglycosylation: Treatment with PNGase F to cleave N-linked glycans from Asn297 in the Fc region of the antibody, which exposes Gln295

• Functional Handle Introduction: Application of microbial transglutaminase (MTGase) to introduce an azide-amine linker at the exposed Gln295 site

• Click Chemistry Conjugation: Conjugation of dibenzocyclooctyne-modified fluorophores (like DBCO-PEG3-FITC) using strain-promoted azide-alkyne cycloaddition

This site-specific approach ensures consistent 1:1 antibody-to-fluorophore ratios and preserves antibody functionality by controlling the conjugation location . For CLIP2 antibodies specifically, this method would ensure that the antigen-binding regions remain unmodified, potentially increasing detection sensitivity in complex neuronal samples where CLIP2 may be expressed at lower levels.

How can researchers optimize CLIP2 Antibody for studying neuronal dendritic lamellar bodies?

For investigating the association between CLIP2 and dendritic lamellar bodies (DLBs), researchers should implement these advanced methodological considerations:

• Sample Preparation: Use fresh or properly fixed neuronal cultures or brain tissue sections with minimal processing to preserve DLB integrity

• Co-localization Studies: Implement dual or triple immunofluorescence labeling using CLIP2 Antibody, FITC conjugated alongside markers for:

  • Microtubules (using anti-tubulin antibodies)

  • Dendritic markers (MAP2)

  • Gap junction proteins (connexins)

• Super-resolution Microscopy: Employ techniques such as STED, STORM, or SIM to resolve the spatial relationship between CLIP2, microtubules, and DLBs beyond the diffraction limit

• Live-cell Imaging: For dynamic studies, consider using membrane-permeable FITC-conjugated antibody fragments to track CLIP2-mediated organelle movements in living neurons

• Functional Disruption: Complement imaging with targeted disruption of CLIP2-microtubule interactions using domain-specific competitors or genetic manipulation

This combined approach allows researchers to not only visualize but also functionally characterize the role of CLIP2 in DLB positioning and function within neuronal dendrites.

What experimental designs best address CLIP2's role in Williams-Beuren syndrome?

Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder associated with a hemizygous deletion of chromosome 7q11.23, which includes the CLIP2 gene . To investigate CLIP2's contribution to WBS pathology, researchers should consider:

• Patient-derived Samples: Compare CLIP2 expression levels in neuronal cells derived from WBS patients versus controls using the FITC-conjugated antibody for quantitative immunofluorescence

• Animal Models: Utilize CLIP2 knockout or hemizygous mouse models to assess:

  • Dendritic morphology alterations

  • Synaptic transmission characteristics

  • Behavioral phenotypes corresponding to WBS symptoms

• Cellular Models: Implement CRISPR-Cas9 mediated CLIP2 deletion or haploinsufficiency in human iPSC-derived neurons to study:

  • Microtubule dynamics and stability

  • Dendritic lamellar body formation and distribution

  • Organelle trafficking along dendrites

• Rescue Experiments: Test whether reintroduction of CLIP2 can rescue cellular phenotypes, using the FITC-conjugated antibody to confirm expression levels

These experimental designs provide complementary approaches to understand CLIP2's mechanistic role in WBS pathophysiology and potential therapeutic interventions.

What common technical issues arise with CLIP2 Antibody, FITC conjugated and how can they be resolved?

When acquiring and analyzing fluorescence data for CLIP2, researchers should normalize signals against appropriate housekeeping proteins and implement quantitative image analysis with defined intensity thresholds to ensure reproducible results across experiments.

How should researchers validate CLIP2 Antibody specificity in different experimental contexts?

Comprehensive validation of CLIP2 Antibody, FITC conjugated should follow these methodological steps:

• Western Blot Confirmation: Despite being FITC-conjugated (typically used for fluorescence applications), perform Western blot analysis to confirm:

  • Single band at the expected molecular weight (~115 kDa)

  • Absence of bands in negative control tissues

• Genetic Validation:

  • Test antibody reactivity in CLIP2 knockout/knockdown cells

  • Observe corresponding decrease in signal intensity

• Epitope Blocking:

  • Pre-incubate antibody with excess immunizing peptide (101-217AA)

  • Verify signal elimination in subsequent staining

• Orthogonal Validation:

  • Compare staining pattern with alternative CLIP2 antibodies recognizing different epitopes

  • Correlate protein detection with mRNA expression (using RNAscope or in situ hybridization)

• Tissue Distribution Analysis:

  • Confirm detection pattern matches known CLIP2 expression profile

  • Verify enrichment in neuronal tissues, particularly in dendritic regions

These validation approaches ensure that experimental observations truly reflect CLIP2 biology rather than antibody artifacts.

How does current research characterize CLIP2's interaction with the microtubule cytoskeleton?

Current research indicates that CLIP2 contains CAP-Gly domains that specifically recognize and bind to microtubule plus-ends. The interaction between CLIP2 and microtubules is particularly critical in neurons, where it appears to:

• Stabilize Microtubules: CLIP2 contributes to microtubule stabilization in dendrites, potentially playing a role in maintaining dendritic architecture

• Facilitate Cargo Transport: By linking microtubules to dendritic lamellar bodies, CLIP2 may create docking stations for cargo delivery at specific dendritic locations

• Regulate Dynamics: CLIP2's association with microtubule plus-ends suggests roles in regulating microtubule growth and shrinkage cycles

The FITC-conjugated CLIP2 antibody enables direct visualization of these interactions in fixed and potentially live-cell imaging studies when combined with tubulin markers. Such dual-labeling approaches have revealed that CLIP2 distribution often shows punctate patterns that coincide with specialized domains of dendritic microtubules.

What innovative conjugation strategies are emerging for antibody-based research tools?

Recent advances in antibody conjugation technologies offer significant advantages for research applications. Site-specific conjugation methods demonstrate particular promise:

• Enzymatic Approaches: The two-step enzymatic method involving PNGase F deglycosylation followed by MTGase-mediated azide handle introduction represents a significant advance over random conjugation methods

• Click Chemistry Applications: Strain-promoted azide-alkyne cycloaddition enables controlled conjugation of antibodies to various research tools including:

  • Fluorophores for imaging (including FITC)

  • Nanoparticles for delivery

  • Virus-based particles for targeting

• Advantages Over Traditional Methods:

  • Preserves antibody functionality by avoiding modification of binding domains

  • Creates homogeneous conjugates with defined stoichiometry (typically 1:1)

  • Prevents aggregation issues common with less-specific conjugation approaches

These methodological advances could be applied to CLIP2 antibodies to create precisely engineered research tools for studying neuronal biology and Williams-Beuren syndrome pathophysiology with increased specificity and reduced background.