KIAA0319 Antibody, FITC conjugated

What is KIAA0319 and why is it significant in neurological research?

KIAA0319 and its paralog KIAA0319L are proteins encoded by genes associated with developmental dyslexia. KIAA0319L is located near the dyslexia susceptibility locus DYX8 in chromosome 1p34.3 and has been suggested as a positional candidate for developmental dyslexia due to its homology with KIAA0319 . Previous research has shown associations between specific markers (e.g., rs7523017) and developmental dyslexia . These proteins are of particular interest because they appear to participate in neuronal development processes including axon guidance, making them important targets for researchers studying neurodevelopmental disorders.

What are the key differences between KIAA0319 and KIAA0319L proteins?

KIAA0319L (also known as AAVR, AAVRL, or AAV receptor) has an expected molecular mass of 115.7 kDa with three reported protein isoforms . In contrast to the primary KIAA0319 protein, KIAA0319L has been found to exist in three distinct forms: two N-glycosylated isoforms (isoforms a and b) and one non-N-glycosylated isoform (isoform c), with a major band appearing at approximately 140 kDa in overexpression studies . Functionally, KIAA0319L has been demonstrated to interact with Nogo Receptor 1 (NgR1), an axon guidance receptor, predominantly in cytoplasmic granules of cortical neurons .

What are the common applications for KIAA0319 antibodies?

KIAA0319 antibodies are commonly used in several research applications:

• Western Blot (WB) - For protein detection and quantification

• Immunohistochemistry (IHC) - Both frozen and paraffin-embedded sections

• Immunohistochemistry-Paraffin (IHC-P) - With recommended dilutions of 1:10 - 1:500

• Peptide ELISA - With detection limits as sensitive as 1:128000

• Immunofluorescence (IF) - Particularly with conjugated antibodies

• Co-immunoprecipitation - For studying protein-protein interactions

For FITC-conjugated variants specifically, the primary applications focus on fluorescence microscopy, flow cytometry, and certain ELISA formats .

How should I optimize protocols for FITC-conjugated KIAA0319 antibodies in immunofluorescence studies?

When designing immunofluorescence experiments with FITC-conjugated KIAA0319 antibodies:

• Fixation method: Paraformaldehyde (4%) is generally recommended for preserving both protein structure and fluorescence signal.

• Dilution optimization: Start with manufacturer's recommendations (typically in the range of 1:10 - 1:500 for unconjugated antibodies) . For FITC-conjugated antibodies, begin with more concentrated dilutions (1:10 - 1:100) and optimize based on signal-to-noise ratio.

• Blocking conditions: Use 5-10% normal serum from the species in which the secondary antibody was raised (not needed for directly conjugated antibodies, but helpful for reducing background).

• Antigen retrieval: For brain tissue sections, citrate buffer (pH 6.0) heat-mediated antigen retrieval often improves signal, particularly for paraffin-embedded samples.

• Controls: Include both positive controls (known expressing tissue) and negative controls (either secondary-only or isotype controls).

What controls are essential when working with FITC-conjugated KIAA0319 antibodies?

Research has shown that appropriate controls are crucial, as an additional band of unknown identity at 25 kDa has been consistently observed in western blots, which can be successfully blocked by incubation with the immunizing peptide .

How should samples be prepared to maximize signal detection with FITC-conjugated antibodies?

For optimal results with FITC-conjugated KIAA0319 antibodies:

• Fresh tissue preparation: Fix tissues immediately after collection to preserve epitope integrity.

• Section thickness: For brain tissue, 5-10 μm sections typically provide optimal results.

• Permeabilization: When studying intracellular domains, use 0.1-0.3% Triton X-100 as used in related KIAA0319L studies .

• Antigen retrieval optimization: KIAA0319 epitopes may be masked in formalin-fixed tissues; test both heat-induced (citrate buffer) and enzymatic methods.

• Photoprotection: FITC is susceptible to photobleaching; minimize exposure to light during all steps and mount with anti-fade reagents containing DAPI for nuclear counterstaining.

• Buffer selection: PBS with neutral pH (7.2-7.4) is recommended based on protocols used in published KIAA0319 studies.

How can I use FITC-conjugated KIAA0319 antibodies to study protein-protein interactions?

To investigate protein-protein interactions involving KIAA0319:

• Co-localization studies: Use FITC-conjugated KIAA0319 antibodies along with differently labeled antibodies against potential binding partners like NgR1, which has been demonstrated to interact with KIAA0319L .

• Live-cell imaging: For real-time interaction studies, FITC-conjugated antibodies can be used with membrane-permeable variants or in permeabilized cells.

• Proximity ligation assay (PLA): Combine FITC-conjugated KIAA0319 antibodies with complementary PLA probes for highly sensitive detection of protein interactions.

• FRET analysis: When combined with appropriately labeled binding partner antibodies, FITC-conjugated antibodies can serve as donor fluorophores in FRET experiments.

• Pull-down confirmation: Follow visualization studies with co-immunoprecipitation experiments similar to those used to confirm KIAA0319L-NgR1 interactions .

Previous research employed both yeast two-hybrid screening and co-immunoprecipitation assays to demonstrate that KIAA0319L and NgR1 physically interact, predominantly in the cytoplasmic granules of cortical neurons in human brain cortex .

What methodologies enable study of KIAA0319 in neural development and axon guidance?

Based on findings that KIAA0319L interacts with NgR1, an axon guidance receptor , researchers can:

• Neuronal culture visualization: Use FITC-conjugated KIAA0319 antibodies to track protein localization during different stages of neuronal differentiation.

• Growth cone analysis: Conduct high-resolution imaging of growth cones to assess KIAA0319 distribution during axon pathfinding.

• Stripe assay analysis: Combine with chemorepulsive or chemoattractive substrates to study how KIAA0319 expression correlates with axon guidance decisions.

• Time-lapse studies: Monitor dynamic changes in KIAA0319 localization during active axon growth and guidance.

• Knockdown studies: Compare KIAA0319 distribution in normal versus siRNA-treated neurons to assess functional consequences.

The interaction between KIAA0319L and NgR1 in cytoplasmic granules of cortical neurons suggests involvement in axon guidance processes, providing a molecular mechanism potentially linking KIAA0319 family proteins to neurodevelopmental processes affected in dyslexia .

How can I address weak or inconsistent signals when using FITC-conjugated KIAA0319 antibodies?

If experiencing weak signals or inconsistent results:

• Antibody concentration adjustment: Increase concentration if signal is weak; commonly used ranges for unconjugated antibodies are 0.5-1.5 μg/ml for Western blot and 1:10-1:500 for immunohistochemistry .

• Epitope accessibility: Ensure the epitope region (for example, C-QGKIKQENKPTLH for some antibodies) is accessible and not masked by fixation or processing .

• Antigen retrieval optimization: If working with paraffin sections, test multiple antigen retrieval methods. Heat-mediated antigen retrieval has been successful in previous KIAA0319 studies .

• Buffer composition: Add protease inhibitors to lysis buffers (as used in RIPA buffer preparations for KIAA0319L studies) .

• Signal amplification: Consider tyramide signal amplification systems if direct FITC conjugates provide insufficient signal.

• Photobleaching prevention: FITC is particularly susceptible to photobleaching; minimize exposure to light during all preparation and imaging steps.

How do I analyze and quantify KIAA0319 expression patterns in brain tissue?

For accurate quantification of KIAA0319 expression:

• Region-specific analysis: Analyze expression separately in different brain regions, as antibody performance may vary across regions with different lipid compositions or protein expression patterns.

• Standardized acquisition: Use consistent exposure settings when acquiring images for comparative analysis.

• Colocalization quantification: When studying interactions with partners like NgR1, use Pearson's correlation coefficient or Manders' overlap coefficient to quantify colocalization.

• Signal normalization: Normalize FITC signal to cell number (using DAPI counterstain) or tissue area for comparing expression levels across samples.

• Automated analysis: Use image analysis software with appropriate thresholding to quantify fluorescence intensity objectively.

Research has shown that KIAA0319L and NgR1 colocalize predominantly in cytoplasmic granules of cortical neurons, requiring careful subcellular localization analysis .

What are the known isoforms of KIAA0319 and how do they affect antibody selection?

KIAA0319L exists in multiple isoforms:

• Isoform diversity: KIAA0319L has three reported protein isoforms . Research has identified two N-glycosylated isoforms (isoform a and isoform b) and one non-N-glycosylated isoform (isoform c) .

• Cross-reactivity: Some antibodies are designed to cross-react with multiple isoforms. For example, certain antibodies are expected to cross-react with mouse KIAA0319 isoform 1 (NP_001074520.1), isoform 2 (XP_994023.1), and isoform 4 (XP_913491.1) .

• Epitope location: The epitope sequence C-QGKIKQENKPTLH corresponds to an internal region of the protein according to Uniprot Mouse NP_001074520.1 . When selecting antibodies, verify that the epitope is present in your isoform of interest.

• Species considerations: Some antibodies show validated reactivity in mouse and predicted reactivity in rat (with 100% sequence homology) .

• Molecular weight verification: When performing Western blots, expect to observe the major band at approximately 140-150 kDa for full-length KIAA0319L, with an additional unidentified band consistently observed at 25 kDa in some preparations .

What is the subcellular localization pattern of KIAA0319 in neuronal cells?

Based on research findings:

• Cytoplasmic granules: KIAA0319L has been observed to interact with NgR1 predominantly in cytoplasmic granules of cortical neurons in human brain cortex .

• Membrane association: As part of the dyslexia-associated protein family, KIAA0319 proteins have membrane-associated domains, though the FITC-conjugated antibodies typically target more accessible regions.

• Developmental regulation: Expression patterns may change during neural development, requiring time-course studies for comprehensive characterization.

• Interaction-dependent localization: The interaction with axon guidance receptors like NgR1 suggests potential association with growth cones and axonal projections during active neurodevelopment .

• Regional variation: Expression and localization patterns may vary across different brain regions, requiring comparative analysis across multiple neural structures.

The colocalization of KIAA0319L with NgR1 in cytoplasmic granules provides evidence supporting the hypothesis that KIAA0319L participates in axon guidance mechanisms, potentially explaining its association with neurodevelopmental disorders like dyslexia .