ANKEF1 Antibody, FITC conjugated

What is ANKEF1 and why is it significant for research?

ANKEF1 (ankyrin repeat and EF-hand domain containing protein 1) is a protein involved in multiple cellular processes, particularly in immune regulation and signaling pathways. Recent evidence indicates a correlation between ANKEF1 expression and cilia formation or function, suggesting its importance in developmental biology . Additionally, ANKEF1 plays a crucial role in protocadherin-mediated cell protrusion and adhesion, with its dysfunction linked to developmental abnormalities in model organisms including spina bifida and shortened body axis in Xenopus studies . The protein's enrichment in ciliated cells and high expression in tissues such as testes and brain make it particularly relevant for research in cellular differentiation, embryonic development, and specialized tissue function . Studying ANKEF1 can provide insights into both normal development and pathological conditions.

What are the specific characteristics of FITC-conjugated ANKEF1 antibodies?

FITC-conjugated ANKEF1 antibodies are immunological tools designed for direct fluorescent detection of ANKEF1 protein. The specific antibody characterized in the search results is a rabbit polyclonal antibody targeting the C-terminal region (amino acids 728 to 757) of ANKEF1 . This antibody demonstrates reactivity with mouse ANKEF1 and is suitable for multiple applications including ELISA and Western blot techniques . The FITC (Fluorescein Isothiocyanate) conjugation provides a direct fluorescent signal with excitation/emission spectra of approximately 495/519 nm, eliminating the need for secondary antibody incubation in fluorescence-based applications. The antibody is supplied in PBS (pH 7.2) containing approximately 0.09% sodium azide . For optimal results, this reagent should be stored in cool conditions but should not be frozen to maintain its immunoreactivity and fluorescent properties.

How does FITC conjugation affect experimental design compared to unconjugated antibodies?

FITC conjugation provides several methodological advantages that influence experimental design:

• Direct detection: FITC-conjugated antibodies enable single-step detection protocols, reducing experimental time and potential sources of background compared to two-step procedures with secondary antibodies .

• Multi-parameter analysis: The FITC fluorophore (emission peak ~519 nm) can be combined with other fluorophores in multi-color flow cytometry or immunofluorescence microscopy for co-localization studies.

• Protocol modifications: Working dilutions for FITC-conjugated antibodies typically differ from unconjugated versions, with recommended dilutions of 1:10-50 for immunochemistry techniques compared to the 1:100-500 range often used for unconjugated antibodies in Western blot applications .

• Photobleaching considerations: Experimental designs must account for FITC's susceptibility to photobleaching, requiring appropriate antifade reagents and minimized exposure to light during storage and handling.

• Autofluorescence interference: When working with tissues known to exhibit autofluorescence in the FITC emission range, additional controls may be necessary compared to experiments using unconjugated antibodies.

The experimental design must balance these factors against the time-saving benefits of direct detection.

What are the validated applications for FITC-conjugated ANKEF1 antibodies and their optimal protocols?

The FITC-conjugated ANKEF1 antibody has been validated for several research applications, each with specific methodological considerations:

*Estimated dilutions based on similar immunochemistry applications

For optimal results in fluorescence-based applications, researchers should:

• Store the antibody at 2-8°C and protect from light

• Include appropriate controls (isotype, negative, and positive)

• Optimize fixation methods based on epitope sensitivity

• Consider tissue/cell-specific autofluorescence when interpreting results

• Never freeze the FITC-conjugated antibody as this may compromise functionality

How can ANKEF1 antibodies be utilized to study its role in cilia formation and function?

Given the correlation between ANKEF1 expression and cilia formation or function , researchers can employ several methodological approaches using FITC-conjugated ANKEF1 antibodies:

• Co-localization studies: Combine FITC-ANKEF1 antibody with markers of ciliary structures (e.g., acetylated tubulin) using confocal microscopy to determine precise subcellular localization.

• Developmental time-course analysis: Apply the antibody at different developmental stages in model organisms to track ANKEF1 expression in relation to ciliogenesis, particularly in dorsal forerunner cells (DFCs) and otic vesicles where the protein has shown discrete expression .

• Comparative expression analysis: Utilize the antibody in diverse ciliated tissues to quantify relative ANKEF1 levels, comparing motile versus primary cilia to establish functional correlations.

• Functional knockdown studies: Combine antibody staining with genetic manipulation techniques (e.g., using ANKEF1 knockout cell lines ) to determine how ANKEF1 depletion affects cilia structure, motility, and signaling functions.

• Flow cytometry of ciliated cells: Use FITC-ANKEF1 antibody in flow cytometry to isolate ciliated cell populations for further molecular characterization.

These approaches can be particularly valuable in tissues where ANKEF1 has shown enriched expression, such as the developing pharynx, swim bladder, testes, and brain .

What methodological considerations are important when using FITC-conjugated antibodies in live cell imaging?

When using FITC-conjugated ANKEF1 antibodies for live cell imaging, researchers must address several methodological challenges:

• Cell membrane permeability: Standard FITC-conjugated antibodies cannot penetrate intact cell membranes. For intracellular targets like ANKEF1, researchers must either:

  • Use membrane permeabilization techniques compatible with cell viability

  • Develop cell-penetrating antibody derivatives

  • Focus on detecting secreted or cell-surface forms of the protein

• Phototoxicity management: FITC excitation can generate reactive oxygen species that damage live cells. Mitigating strategies include:

  • Minimizing exposure time and light intensity

  • Using antioxidant supplements in imaging media

  • Employing oxygen scavenger systems

  • Utilizing advanced imaging techniques like spinning disk confocal microscopy

• Signal stability: Since FITC is susceptible to photobleaching, researchers should:

  • Optimize acquisition parameters to minimize total light exposure

  • Consider time-lapse imaging intervals carefully

  • Use computational approaches to correct for signal decay

• Physiological relevance: Antibody binding may alter protein function. Controls should include:

  • Functional assays to assess potential interference with normal ANKEF1 activity

  • Comparison with alternative labeling strategies (e.g., fluorescent protein fusions)

• Environmental sensitivity: FITC fluorescence can be affected by pH variations common in live cell experiments, requiring careful experimental design and calibration .

How can researchers address non-specific binding and background issues with FITC-conjugated ANKEF1 antibodies?

Non-specific binding and background fluorescence are common challenges when working with FITC-conjugated antibodies, including those targeting ANKEF1. Here are methodological approaches to overcome these issues:

• Optimizing blocking conditions:

  • Use a protein blocker appropriate for your sample type (e.g., 5% BSA, 10% normal serum from the same species as secondary antibody)

  • Consider specialized blocking reagents for tissues with high endogenous biotin or charged proteins

  • Extend blocking time to 2 hours at room temperature for problematic samples

• Antibody titration:

  • Perform systematic dilution series (e.g., 1:10, 1:50, 1:100, 1:500) to identify optimal signal-to-noise ratio

  • For Western blotting, keep dilutions within the recommended 1:100-500 range

  • For immunochemistry, start at the recommended 1:10-50 dilution and adjust based on results

• Managing autofluorescence:

  • For tissues with high autofluorescence (e.g., brain, kidney), pretreat with Sudan Black B (0.1-0.3%)

  • Use spectral unmixing on confocal systems capable of lambda scanning

  • Consider alternative fluorophores if FITC's emission spectrum significantly overlaps with sample autofluorescence

• Specialized controls:

  • Include isotype controls (FITC-conjugated rabbit IgG) at equivalent concentrations

  • Use ANKEF1 knockout cells as negative controls to confirm antibody specificity

  • Perform peptide competition assays using the immunizing peptide (amino acids 728-757)

• Buffer optimization:

  • Adjust detergent concentration in wash buffers (typically 0.05-0.1% Tween-20)

  • Consider adding 0.1-0.3M NaCl to reduce electrostatic interactions

  • Maintain proper pH (7.2-7.4) in all solutions to ensure optimal FITC fluorescence

Implementation of these approaches should be systematic, changing only one variable at a time to identify the specific factors contributing to background issues.

What are the most common technical pitfalls when working with ANKEF1 antibodies in different model systems?

Working with ANKEF1 antibodies across different model systems presents several technical challenges that researchers should anticipate:

• Species cross-reactivity limitations:

  • The FITC-conjugated ANKEF1 antibody described shows reactivity with mouse ANKEF1 , while other ANKEF1 antibodies may be specific to human samples .

  • When working with zebrafish, researchers must consider the presence of two paralogs (ankef1a and ankef1b) , potentially requiring paralog-specific antibodies.

  • Cross-reactivity testing is essential when applying antibodies to non-validated species.

• Epitope accessibility issues:

  • The C-terminal epitope (amino acids 728-757) targeted by the FITC-conjugated antibody may have differential accessibility in various experimental contexts.

  • Fixation methods can significantly impact epitope recognition; paraformaldehyde may mask the epitope while methanol fixation might preserve it.

  • Alternative epitope antibodies should be considered if consistent detection problems occur.

• Developmental stage-specific considerations:

  • ANKEF1 expression patterns change during development , requiring careful timing of experiments.

  • For embryonic studies, background autofluorescence from yolk proteins may interfere with FITC detection.

  • Tissue-specific expression patterns (e.g., enrichment in testes, brain, pharynx, and swim bladder) necessitate tailored approaches for different organ systems.

• Technical compatibility issues:

  • FITC's sensitivity to photobleaching requires special handling during long imaging sessions.

  • The antibody's storage conditions (cool but not frozen) may complicate field research logistics.

  • The limited compatibility with certain fixatives may affect multi-labeling experimental designs.

• Validation challenges:

  • Given ANKEF1's emerging research status, positive control samples with confirmed expression may be difficult to obtain.

  • Using ANKEF1 knockout cell lines as negative controls is recommended for definitive validation.

Researchers should document these variables meticulously and consider preliminary validation experiments before proceeding with larger studies.

How can researchers validate the specificity of ANKEF1 antibody binding in experimental systems?

• Genetic validation approaches:

  • Utilize ANKEF1 knockout cell lines as negative controls to confirm absence of signal

  • Perform siRNA/shRNA knockdown experiments with quantitative analysis of signal reduction

  • Compare staining patterns in wild-type versus ANKEF1-overexpressing systems

• Biochemical validation methods:

  • Conduct peptide competition assays using the immunizing peptide (amino acids 728-757)

  • Perform immunoprecipitation followed by mass spectrometry to confirm target identity

  • Compare results from antibodies targeting different ANKEF1 epitopes

• Technical validation approaches:

  • Compare localization patterns with published data on ANKEF1 distribution

  • Verify that expression patterns match known tissue-specific distributions (e.g., enrichment in testes, brain)

  • Confirm co-localization with known interacting partners or subcellular structures

• Multi-method confirmation:

  • Validate protein detection using orthogonal techniques (e.g., Western blot, immunofluorescence, flow cytometry)

  • Compare protein expression with mRNA expression data from RT-qPCR or RNA-seq

  • Confirm functionality through rescue experiments in knockout/knockdown systems

• Specificity controls in multiplex experiments:

  • Include single-stain controls when combining with other fluorescent probes

  • Verify absence of unexpected cross-reactivity with other proteins by examining signal in tissues known to lack ANKEF1 expression

A validation matrix documenting these approaches should be maintained for each new experimental system or application to ensure reproducible research outcomes.

How can FITC-conjugated ANKEF1 antibodies be integrated into multi-parameter flow cytometry panels?

Integrating FITC-conjugated ANKEF1 antibodies into multi-parameter flow cytometry panels requires strategic planning to maximize data quality and analytical potential:

• Panel design considerations:

  • FITC's emission spectrum (peak ~519 nm) requires careful selection of compatible fluorophores to minimize spectral overlap

  • Recommended fluorophore combinations with FITC include PE (phycoerythrin), APC (allophycocyanin), and BV421 (Brilliant Violet 421)

  • Reserve FITC for moderate-to-high abundance targets like ANKEF1 in ciliated or highly expressing cells (e.g., testes, brain)

• Optimization protocol:

  • Begin with titration experiments using the recommended dilution range (starting at 1:50)

  • Determine optimal voltage settings for the FITC channel using single-stained controls

  • Establish compensation matrix using single-color controls for each fluorophore

  • Include Fluorescence Minus One (FMO) controls to set accurate gating boundaries

• Sample preparation considerations:

  • For intracellular ANKEF1 detection, optimize fixation and permeabilization conditions that maintain epitope integrity

  • Use viability dyes compatible with FITC to exclude dead cells

  • When analyzing ciliated cells, consider special dissociation protocols that preserve ciliary structures

• Analytical approach:

  • Develop gating strategies that incorporate known ANKEF1 expression patterns in specific cell populations

  • Consider using dimensionality reduction algorithms (e.g., tSNE, UMAP) for exploratory analysis of ANKEF1 co-expression patterns

  • Correlate ANKEF1 expression with functional parameters or other protein markers

• Data validation strategy:

  • Compare flow cytometry results with quantitative imaging or Western blot data

  • Validate findings using ANKEF1 knockout cells as negative controls

  • Confirm specificity through isotype control staining at equivalent concentrations

This methodical approach allows researchers to generate reliable multi-parameter data on ANKEF1 expression in heterogeneous cell populations.

What are the considerations for using ANKEF1 antibodies in studying its role in embryonic development and ciliated tissues?

Studying ANKEF1's role in embryonic development and ciliated tissues requires tailored experimental approaches that address the protein's temporal and spatial expression patterns:

• Developmental timing considerations:

  • ANKEF1 expression is dynamic during development, with ubiquitous expression early in embryogenesis followed by more discrete patterns

  • Experimental timepoints should capture key developmental transitions, particularly during ciliogenesis

  • For zebrafish studies, examine expression in dorsal forerunner cells (DFC) and otic vesicles, which precede or possess motile cilia

• Tissue-specific methodological adaptations:

  • For testes, where ANKEF1 shows high expression , optimize fixation to preserve both structural integrity and epitope accessibility

  • In brain tissue, implement autofluorescence reduction strategies to enhance FITC signal detection

  • For pharynx and swim bladder analysis, consider tissue clearing techniques to improve imaging depth

• Functional assessment approaches:

  • Combine FITC-ANKEF1 antibody staining with markers of ciliary function (e.g., acetylated tubulin)

  • Correlate ANKEF1 expression with phenotypic outcomes observed in knockdown models (e.g., shortened body axis, spina bifida)

  • Implement live imaging protocols to track dynamic processes in ciliated tissues

• Comparative model systems analysis:

  • Consider paralog-specific approaches when working with zebrafish (ankef1a and ankef1b)

  • When transitioning between model systems, validate antibody cross-reactivity thoroughly

  • Implement consistent quantification methods to compare expression levels across different developmental stages and organisms

• Signaling pathway integration:

  • Investigate ANKEF1's relationship with FGF signaling, given evidence of downregulation after FGF inhibition

  • Explore connections to protocadherin-mediated processes in cell protrusion and adhesion

  • Consider potential relationships with other signaling pathways involved in ciliogenesis

These methodological considerations enable researchers to generate more comprehensive insights into ANKEF1's developmental functions and ciliary relationships.

How do expression levels and patterns of ANKEF1 vary across tissues, and what implications does this have for immunodetection strategies?

ANKEF1 exhibits distinct expression patterns across tissues, necessitating tailored immunodetection strategies for accurate analysis:

These expression variations have several implications for immunodetection strategies:

• Concentration and dilution optimization:

  • Tissues with high expression (testes, brain) may require higher antibody dilutions (1:300-500) to prevent oversaturation

  • Areas with discrete expression may need lower dilutions (1:10-50) as recommended

  • Titration experiments should be performed for each tissue type

• Tissue-specific fixation protocols:

  • Ciliated structures require gentle fixation methods that preserve delicate morphology

  • Brain tissue benefits from specialized fixation protocols that address lipid content

  • Embryonic tissues may require different fixation parameters than adult samples

• Background mitigation strategies:

  • Tissues with high autofluorescence (brain, swim bladder) require specific countermeasures

  • Complex tissues may benefit from longer blocking steps with specialized blocking agents

  • Signal amplification methods may be needed for tissues with lower expression

• Imaging parameter adjustments:

  • Exposure and gain settings should be optimized for different expression levels

  • Z-stack parameters must account for tissue-specific ANKEF1 distribution patterns

  • Resolution requirements vary based on subcellular localization patterns

• Quantification approaches:

  • Relative quantification methods should account for tissue-specific expression baselines

  • Internal controls specific to each tissue type enhance quantitative reliability

  • When comparing across tissues, normalization strategies must address inherent expression differences

Understanding these tissue-specific variations is crucial for developing robust, reproducible immunodetection protocols for ANKEF1 research.

What are the emerging research areas where ANKEF1 antibodies may prove valuable?

Several emerging research areas show potential for significant advances through application of ANKEF1 antibodies:

• Ciliopathy mechanisms:

  • The correlation between ANKEF1 expression and cilia formation/function suggests potential roles in primary ciliary dyskinesia, Bardet-Biedl syndrome, and related disorders

  • ANKEF1 antibodies could help establish diagnostic biomarkers for ciliopathies

  • Screening for ANKEF1 dysregulation may identify novel disease mechanisms

• Developmental biology:

  • Given ANKEF1's critical role in embryonic development and the severe phenotypes observed in morphants (spina bifida, shortened body axis) , antibodies could help map developmental signaling networks

  • The discrete expression in specialized embryonic structures suggests tissue-specific developmental functions awaiting characterization

  • ANKEF1's relationship with FGF signaling warrants further investigation in developmental contexts

• Reproductive biology:

  • High expression in testes points to potential roles in spermatogenesis or sperm function

  • Potential connections to fertility disorders could be explored through immunodetection in clinical samples

  • The relationship between ANKEF1 and ciliary structures suggests possible functions in sperm motility

• Neuroscience applications:

  • Enriched brain expression indicates neurobiological functions that remain largely unexplored

  • Primary cilia in neurons have emerging roles in neurodevelopment and signaling

  • ANKEF1 antibodies could help characterize protein distribution across neural cell types and brain regions

• Cellular adhesion and migration:

  • ANKEF1's role in protocadherin-mediated cell protrusion and adhesion suggests applications in cancer metastasis research

  • Antibodies could help characterize ANKEF1's contributions to cell-cell interactions in various contexts

  • The protein's relationship to ankyrin repeat domains, known for protein-protein interactions, warrants exploration in cellular signaling networks

These emerging areas represent fertile ground for innovative research applications of FITC-conjugated and other ANKEF1 antibodies.

How can ANKEF1 antibodies be combined with advanced imaging techniques to gain new insights into protein function?

Combining ANKEF1 antibodies with cutting-edge imaging approaches can reveal previously unattainable insights into protein function and dynamics:

• Super-resolution microscopy applications:

  • STED (Stimulated Emission Depletion) microscopy can resolve ANKEF1 localization at sub-diffraction resolutions (~30-80 nm), revealing precise ciliary distribution patterns

  • STORM/PALM techniques can map individual ANKEF1 molecules within cellular structures, potentially identifying clustering patterns or isolation within membrane microdomains

  • Structured Illumination Microscopy (SIM) offers enhanced resolution with less photobleaching than other super-resolution techniques, beneficial for FITC-conjugated antibodies

• Live-cell dynamics approaches:

  • For surface-accessible forms of ANKEF1, Fab fragments of FITC-conjugated antibodies could enable live tracking with reduced interference

  • Correlative Light and Electron Microscopy (CLEM) can connect ANKEF1 fluorescence patterns with ultrastructural features

  • Fluorescence Recovery After Photobleaching (FRAP) with carefully designed cell-permeable antibody derivatives could assess ANKEF1 mobility

• Multiplexed detection strategies:

  • Imaging Mass Cytometry combines ANKEF1 antibody detection with measurement of dozens of additional proteins in the same sample

  • Cyclic immunofluorescence (CycIF) enables sequential staining rounds to correlate ANKEF1 with numerous markers beyond what conventional fluorescence allows

  • Spatial transcriptomics combined with ANKEF1 immunodetection can correlate protein expression with transcriptional profiles at tissue scale

• Functional imaging integration:

  • Optogenetic manipulation combined with ANKEF1 immunodetection can reveal dynamic responses to controlled signaling perturbations

  • Calcium imaging paired with ANKEF1 staining in fixed samples can correlate functional activity with protein distribution

  • FRET-based approaches using appropriately labeled antibody pairs could potentially detect ANKEF1 conformational changes or protein-protein interactions

• Tissue-scale approaches:

  • Light-sheet microscopy enables rapid, low-phototoxicity imaging of ANKEF1 distribution across large tissue volumes

  • Tissue clearing techniques (CLARITY, CUBIC, iDISCO) combined with ANKEF1 antibodies allow deep tissue imaging in intact organs

  • Expansion microscopy physically enlarges samples, providing enhanced resolution of ANKEF1 localization using standard confocal microscopy

These methodological combinations can significantly advance understanding of ANKEF1's localization, dynamics, and functional relationships within complex biological systems.