ANKEF1 Antibody

What is the optimal ANKEF1 antibody type for detecting human samples?

For human ANKEF1 detection, rabbit polyclonal antibodies are widely available and validated. These antibodies typically target recombinant human ANKEF1 protein, particularly the C-terminal region (411-698 AA) . When selecting an antibody, consider:

• Polyclonal antibodies offer broader epitope recognition, enhancing detection sensitivity

• Confirm human reactivity in product specifications

• The UniProt ID for human ANKEF1 is Q9NU02, which can be cross-referenced when selecting antibodies

• Available conjugations include HRP, FITC, and PE, allowing flexibility in experimental design based on detection method

How does antibody conjugation affect ANKEF1 detection methodology?

Different conjugations enable specific detection methodologies:

Selection should be based on your specific experimental approach and available detection instruments .

What are the optimal conditions for ANKEF1 antibody storage and handling?

Proper storage and handling are critical for maintaining antibody functionality:

• Store aliquoted antibody at -20°C to minimize freeze-thaw damage

• Avoid repeated freeze/thaw cycles as this significantly reduces antibody activity

• PE-conjugated antibodies are particularly sensitive; product documentation often states "Do not freeze!"

• Most commercially available ANKEF1 antibodies are supplied in a buffer containing 0.01M PBS (pH 7.4), 0.03% Proclin-300, and 50% glycerol

• Working dilutions should be prepared fresh and used within 24 hours for optimal results

What dilutions are recommended for different ANKEF1 antibody applications?

Application-specific dilutions vary based on conjugation and experimental context:

Always perform a dilution series during optimization, as "optimal dilutions/concentrations should be determined by the end user" .

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

ANKEF1 has been correlated with cilia formation and function in recent NGS studies . Research methodologies include:

• Co-localization studies using ANKEF1 antibodies with established ciliary markers

• Immunofluorescence microscopy of ciliated tissues with FITC-conjugated ANKEF1 antibodies

• Proteomic analysis has shown ANKEF1 enrichment in ciliated cells

• For developmental studies, focus on tissues known to precede or possess motile cilia, such as dorsal forerunner cells (DFC) and otic vesicles

• Knockdown studies followed by phenotypic analysis and rescue experiments can establish functional relationships

In zebrafish models, ankef1a and ankef1b transcripts were detected in embryonic tissues with motile cilia, supporting their role in ciliary development or function .

What considerations are important when studying ANKEF1 across different vertebrate species?

ANKEF1 research across species reveals important evolutionary and functional insights:

• Human and mouse ANKEF1 antibodies may have varied cross-reactivity; verify species reactivity in specifications

• Zebrafish have two paralogs (ankef1a and ankef1b) that should be distinguished in experimental design

• In Xenopus laevis, ankef1 is downregulated after FGF inhibition, suggesting different regulatory mechanisms across species

• Sequence homology analysis shows mouse ANKEF1 has 80% identity and rat ANKEF1 has 81% identity to human ANKEF1 in the immunogen sequence region

• When designing cross-species experiments, consider using conserved epitopes for antibody selection

Zebrafish model systems are particularly valuable for developmental studies, as both ankef1a and ankef1b show enriched expression in developing pharynx and swim bladder .

Which tissues show highest ANKEF1 expression, and how should antibody-based detection be optimized for these tissues?

ANKEF1 shows tissue-specific expression patterns that inform experimental design:

• EST profile databases indicate expression in multiple human tissues, with notably high levels in testes

• qPCR results from zebrafish models show enhanced expression in testes and brain

• For high-expressing tissues, antibody dilutions can be adjusted to 1:300-500 for Western blot applications

• For low-expressing tissues, consider using signal amplification methods with HRP-conjugated antibodies

• In developmental studies, consider whole-mount in situ hybridization (WISH) as a complementary approach to antibody staining

When studying reproductive or neurological tissues, optimization of fixation protocols is crucial for preserving epitope accessibility while maintaining tissue morphology.

How can ANKEF1 antibodies be employed to investigate its role in embryonic development?

Studies in Xenopus and zebrafish highlight ANKEF1's developmental importance:

• In Xenopus laevis, ankef1 morphants exhibit spina bifida and shortened body axis

• For developmental studies, time-course immunohistochemistry can track spatial and temporal expression

• FITC-conjugated antibodies allow visualization in whole-mount preparations of embryonic tissues

• In zebrafish, both ankef1a and ankef1b are ubiquitously expressed early in development before localizing to specific structures

• When designing developmental experiments, consider co-staining with markers for protocadherin-mediated cell protrusion and adhesion, as ANKEF1 plays a role in these processes

For optimal results in embryonic tissues, titrate antibody concentrations and adjust permeabilization protocols to ensure adequate penetration without compromising epitope integrity.

What are common issues with ANKEF1 antibody specificity, and how can they be addressed?

Addressing specificity concerns requires systematic validation:

• Verify antibody specificity using positive and negative controls (tissues/cells known to express or lack ANKEF1)

• Consider using knockout/knockdown models as gold-standard negative controls

• For polyclonal antibodies, lot-to-lot variation may occur; maintain consistent lot numbers for longitudinal studies

• Pre-absorption with immunizing peptide can confirm specificity in challenging applications

• Western blot validation should confirm detection at the expected molecular weight before proceeding to other applications

If cross-reactivity is observed, increasing antibody dilution and optimizing washing steps can improve specificity without compromising sensitivity.

How can researchers optimize immunoprecipitation protocols for ANKEF1 interaction studies?

Immunoprecipitation (IP) optimization for protein interaction studies:

• Select antibodies raised against epitopes unlikely to be masked by protein interactions

• For co-IP experiments, gentler lysis buffers (containing 0.5-1% NP-40 or Triton X-100) help preserve protein-protein interactions

• Pre-clear lysates with protein G beads to reduce non-specific binding

• Consider crosslinking the antibody to beads to prevent antibody contamination in the eluted sample

• For detecting weak or transient interactions, proximity ligation assays using FITC-conjugated ANKEF1 antibodies may be more sensitive than traditional co-IP

When studying ANKEF1's role in protocadherin-mediated cell adhesion, co-IP followed by mass spectrometry can identify novel interaction partners within adhesion complexes.

How can ANKEF1 antibodies be utilized in studying its potential roles in disease states?

While direct disease associations remain under investigation, several approaches can be employed:

• Compare ANKEF1 expression in normal versus pathological tissues, particularly in ciliopathies

• Investigate ANKEF1 localization changes during disease progression using immunofluorescence

• Examine post-translational modifications of ANKEF1 in disease states using modification-specific antibodies

• For functional studies, combine antibody detection with genetic manipulation (CRISPR, RNAi)

• Consider multiple detection methods (Western blot, IHC, flow cytometry) for comprehensive analysis

Given the enrichment of ANKEF1 in ciliated cells, particular focus should be placed on diseases involving ciliary dysfunction, including primary ciliary dyskinesia and polycystic kidney disease.

What considerations are important when designing multiplexed detection systems incorporating ANKEF1 antibodies?

Multiplexed detection requires careful planning:

• When selecting ANKEF1 antibodies for multiplexing, consider spectral compatibility of conjugates:

  • FITC (Ex/Em: 499/515 nm) pairs well with red-shifted fluors

  • PE-conjugated antibodies offer brightness but may overlap with other commonly used fluorophores

• Host species must differ between primary antibodies to avoid cross-reactivity

• Sequential immunostaining protocols may be necessary if antibodies are from the same host species

• Include appropriate controls to assess and correct for spectral overlap

• For flow cytometry applications, perform compensation controls using single-stained samples

When investigating ANKEF1's relationship with ciliary structures, co-staining with established ciliary markers provides valuable context for functional studies.