ANKRD20A3 Antibody

What is ANKRD20A3 and why is it important for research?

ANKRD20A3 is a member of the ankyrin repeat domain protein family. Ankyrin proteins contain multiple ankyrin repeats that mediate protein-protein interactions and are crucial for various cellular functions. Mutations in ankyrin genes are associated with severe genetic diseases, including fatal cardiac arrhythmias and hereditary spherocytosis . Understanding ANKRD20A3's function through antibody-based detection methods provides insights into its biological roles and potential involvement in pathological conditions.

What types of ANKRD20A3 antibodies are available for experimental applications?

Several types of ANKRD20A3 antibodies are available, each optimized for specific applications:

The conjugated variants include antibodies labeled with AbBy Fluor® 488, 555, 594, 647, 680, and Cy3 for immunofluorescence applications .

How do I determine the appropriate experimental controls when using ANKRD20A3 antibodies?

For rigorous experimental design with ANKRD20A3 antibodies, implement these controls:

• Positive tissue/cell control: Use samples known to express ANKRD20A3 (human cell lines have demonstrated reactivity in most studies)

• Negative control:

  • Primary antibody omission

  • Non-immune serum from the same species

  • Tissues/cells known not to express the target

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (synthetic peptide derived from human ANKRD20A3) to confirm specificity

• Knockdown/knockout validation: If possible, use ANKRD20A3 knockdown or knockout samples to validate antibody specificity

What are the optimized protocols for immunohistochemistry using ANKRD20A3 antibodies?

For optimal immunohistochemical detection of ANKRD20A3:

• Sample preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin

  • Section at 4-6μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Pressure cooker treatment for 10-15 minutes recommended

• Antibody incubation:

  • Dilution ranges: 1:50-1:200 for IHC applications

  • Incubate at 4°C overnight or at room temperature for 1-2 hours

  • Use a humidity chamber to prevent section drying

• Detection system:

  • HRP-conjugated secondary antibody followed by DAB substrate

  • For fluorescent detection, use appropriate fluorophore-conjugated secondary antibodies

  • Consider biotin-conjugated variants for signal amplification in tissues with low expression

• Counterstaining and mounting:

  • Counterstain with hematoxylin for brightfield or DAPI for fluorescence

  • Mount with appropriate mounting media based on detection method

What are the recommended protocols for Western blot analysis of ANKRD20A3?

For effective Western blot detection of ANKRD20A3:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for protein extraction

  • Expected molecular weight: ~94-95 kDa

• Gel electrophoresis and transfer:

  • 8-10% SDS-PAGE recommended due to protein size

  • Transfer to PVDF membrane at 25V overnight at 4°C

• Antibody incubation:

  • Block with 5% non-fat milk or BSA

  • Primary antibody dilution: 1:500-1:3000 or 1:1000

  • Incubate overnight at 4°C with gentle agitation

• Detection and analysis:

  • Use HRP-conjugated secondary antibody and ECL detection

  • Include molecular weight markers to confirm band size

  • Consider longer exposure times if signal is weak

How should immunofluorescence protocols be optimized for ANKRD20A3 detection?

For immunofluorescence applications:

• Cell preparation:

  • Fix cells with 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilize with 0.1-0.3% Triton X-100 (5-10 minutes)

• Antibody selection and dilution:

  • For paraffin sections (IF-p): Use antibodies verified for this application

  • Recommended dilution: 1:100-1:500 for IF/ICC applications

  • Consider directly conjugated antibodies (AbBy Fluor® 488, 555, 594, 647, 680, Cy3) for multiplexing

• Incubation conditions:

  • Incubate primary antibody overnight at 4°C

  • Wash thoroughly (3-5 times with PBS)

  • Secondary antibody incubation: 1-2 hours at room temperature

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI

  • Mount with anti-fade mounting medium to preserve fluorescence

How can ANKRD20A3 antibodies be validated for specificity and reproducibility?

Comprehensive validation approaches include:

• Multi-method concordance:

  • Compare protein detection across multiple methods (WB, IHC, IF)

  • Consistent molecular weight and localization patterns should be observed

• Epitope mapping:

  • Use antibodies targeting different epitopes (e.g., AA 551-600 and AA 794-821 C-terminus)

  • Consistent results across different epitope-targeted antibodies support specificity

• Recombinant expression:

  • Overexpress tagged ANKRD20A3 in cell lines

  • Confirm colocalization of antibody signal with tag-specific antibodies

• Cross-reactivity assessment:

  • Test reactivity in tissues from different species

  • Most ANKRD20A3 antibodies show primary reactivity to human samples, though some demonstrate cross-reactivity with mouse and rat samples

• Lot-to-lot validation:

  • Compare performance across different antibody lots

  • Document key validation parameters for each lot

What are the most common troubleshooting issues with ANKRD20A3 antibodies?

Key troubleshooting approaches for common issues:

• Weak or no signal:

  • Increase antibody concentration (within recommended ranges)

  • Optimize antigen retrieval methods (extend time or try alternative buffers)

  • Extend incubation time for primary antibody

  • Ensure target protein is present (use positive controls)

  • Check for proper storage conditions (avoid repeated freeze-thaw cycles)

• High background:

  • Increase blocking time/concentration

  • Reduce primary antibody concentration

  • Increase wash steps duration/frequency

  • Use more specific secondary antibodies

  • Prepare fresh buffers to avoid contamination

• Non-specific bands in Western blot:

  • Optimize blocking conditions

  • Increase antibody specificity through affinity purification

  • Adjust sample preparation to minimize protein degradation

  • Consider alternative lysis buffers to improve protein extraction

• Inconsistent staining patterns:

  • Standardize fixation protocols

  • Control incubation temperatures precisely

  • Prepare antibody dilutions fresh before each experiment

  • Use automated staining platforms for greater consistency

How can ANKRD20A3 antibodies be utilized in multiplexed immunofluorescence studies?

For successful multiplexed detection:

• Antibody panel design:

  • Select fluorophore-conjugated ANKRD20A3 antibodies compatible with your microscopy setup

  • Available conjugates include AbBy Fluor® 488, 555, 594, 647, 680, and Cy3

  • Plan panel based on spectral separation and expression levels of targets

• Sequential staining approach:

  • For unconjugated antibodies, use sequential staining with thorough washing

  • Consider tyramide signal amplification for weak signals

  • Test for cross-reactivity between antibodies in the panel

• Image acquisition and analysis:

  • Use appropriate filter sets to minimize spectral overlap

  • Implement spectral unmixing algorithms if necessary

  • Include single-stained controls for each fluorophore

  • Use computational approaches for colocalization analysis

What is known about ANKRD20A3 expression in cancer research models?

ANKRD20A3 has been investigated in cancer research contexts:

• Breast cancer relevance:

  • Studies have examined ANKRD20A3 in the context of metastasized breast cancer cells

  • Potential association with migratory phenotypes in cancer cell lines

• Expression analysis approaches:

  • Principal component analysis (PCA) has been used to analyze ANKRD20A3 expression in cancer cell lines

  • Integration with other genomic data can provide insights into functional pathways

• Methodology for expression studies:

  • 2D and 3D culture systems for investigating protein expression

  • Cell models include:

    • Standard 2D cultures in appropriate media

    • 3D spheroid models using low-adhesion plates

    • Migration and invasion assays for functional assessment

How can ANKRD20A3 antibodies be used for studying protein-protein interactions?

Methodological approaches include:

• Co-immunoprecipitation (Co-IP):

  • Use purified ANKRD20A3 antibodies for pull-down experiments

  • Affinity-purified antibodies provide better specificity for immunoprecipitation

  • Confirm results with reverse Co-IP using antibodies against interacting proteins

• Proximity ligation assay (PLA):

  • Combine ANKRD20A3 antibodies with antibodies against suspected interaction partners

  • This technique allows visualization of protein interactions within 40nm distance

  • Requires optimization of antibody dilutions and incubation conditions

• FRET/FLIM analysis:

  • Use fluorophore-conjugated ANKRD20A3 antibodies for FRET analysis

  • Available conjugates like AbBy Fluor® 488 (donor) and AbBy Fluor® 555/594 (acceptor) are suitable

  • Control experiments essential to validate energy transfer

What are the optimal storage conditions for maintaining ANKRD20A3 antibody activity?

For maximum antibody stability and performance:

• Temperature considerations:

  • Store at -20°C for long-term storage

  • Some preparations may benefit from -80°C storage for extended shelf-life

  • Avoid repeated freeze-thaw cycles that can degrade antibody quality

• Buffer composition:

  • Most ANKRD20A3 antibodies are supplied in phosphate buffered saline (pH 7.4)

  • Buffer typically contains 150mM NaCl, 0.02% sodium azide, and 50% glycerol

  • The glycerol content prevents freezing solid and reduces freeze-thaw damage

• Aliquoting strategy:

  • Prepare small working aliquots upon receipt

  • Use sterile conditions when handling to prevent contamination

  • Document date of thawing and number of freeze-thaw cycles

• Working solution handling:

  • Diluted antibody solutions should be prepared fresh

  • Store working dilutions at 4°C for no more than one week

  • Include stabilizing proteins (BSA) in working dilutions