Recombinant Pongo abelii Ankyrin repeat domain-containing protein 46 (ANKRD46)

What is ANKRD46 and what is its function in Pongo abelii?

ANKRD46 (Ankyrin repeat domain-containing protein 46) is a protein that contains multiple ankyrin repeat domains. These ankyrin domains primarily function in protein-protein interactions across various cellular processes. In Pongo abelii (Sumatran orangutan), ANKRD46 likely plays similar roles to its human ortholog, though species-specific functions may exist .

The molecular structure includes multiple ankyrin repeat motifs which typically form scaffolds for specific protein-protein interactions. These interactions are integral to cellular signaling pathways, though the specific pathways for ANKRD46 in Pongo abelii are not fully characterized in current literature.

How does Pongo abelii ANKRD46 compare structurally with its human ortholog?

The human ANKRD46 gene is located on chromosome 8q22.3 and contains 7 exons . While complete comparative analysis is not explicitly described in the available literature, orthologous proteins between closely related primates typically share high sequence homology.

The functional ankyrin repeat domains are likely conserved between human and Pongo abelii ANKRD46, as these domains are critical for the protein's fundamental interactions. Researchers should consider performing sequence alignment analysis using tools like BLAST to determine percent identity and identify conserved functional domains when designing comparative studies.

What are the recommended storage conditions for recombinant Pongo abelii ANKRD46?

For optimal stability of recombinant Pongo abelii ANKRD46:

• Store at -20°C for regular storage

• For extended storage, conserve at -20°C or -80°C

• Prepare working aliquots and store at 4°C for up to one week

• Avoid repeated freezing and thawing cycles as this may compromise protein integrity

• The protein is typically provided in a Tris-based buffer with 50% glycerol, optimized for this specific protein

How should I design qPCR assays for ANKRD46 expression analysis?

When designing qPCR assays for ANKRD46 expression analysis, follow these methodological guidelines:

• Primer design considerations:

  • Design primers that span exon-exon junctions to prevent genomic DNA amplification

  • Ensure amplicon size is around 100 bases (rather than longer amplicons) for optimal efficiency

  • Check for SNPs in primer binding regions that might affect amplification efficiency

  • Verify primer specificity using BLAST to avoid cross-reactivity with similar sequences

• Reference gene selection:

  • Do not rely solely on traditional reference genes like GAPDH or ACTB without validation

  • Evaluate multiple reference genes for stability in your specific experimental conditions

  • Use a minimum of 2-3 validated reference genes for normalization

• Quality control measures:

  • Include both positive and negative controls

  • Generate a robust standard curve for accurate quantification

  • Completely remove residual RNA from cDNA samples to avoid skewing normalization

  • Follow MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) to ensure reproducibility

What approaches can be used to study protein-protein interactions involving ANKRD46?

As an ankyrin repeat domain-containing protein that functions in protein-protein interactions, several methodological approaches are appropriate for investigating ANKRD46 binding partners:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies against ANKRD46 to pull down the protein along with its binding partners

  • Identify interacting proteins using mass spectrometry

  • Verify interactions with Western blotting for suspected partners

• Yeast two-hybrid screening:

  • Create fusion constructs with ANKRD46 as bait

  • Screen against cDNA libraries from relevant tissues to identify novel interactors

• Proximity-based labeling methods:

  • BioID or APEX2 fusion proteins can identify proteins in close proximity to ANKRD46

  • These approaches capture transient and weak interactions that might be missed by Co-IP

• Recombinant protein interaction assays:

  • Express and purify recombinant ANKRD46

  • Perform pull-down assays with candidate interacting proteins

  • Quantify binding kinetics using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC)

How can I investigate the potential role of ANKRD46 in gene regulatory networks?

Meta-analysis approaches can be applied to investigate ANKRD46's role in gene regulatory networks:

• Matched aCGH/expression analysis in cancer datasets:

  • Utilize copy number variation and gene expression correlation analysis

  • Apply robust statistical methods to identify potential regulatory relationships

  • Use a meta-analysis approach across multiple datasets to strengthen statistical power

  • Implement careful data randomization to generate appropriate null distributions for statistical testing

• Functional validation strategies:

  • Perform knockdown/knockout experiments followed by RNA-seq to identify affected genes

  • Create domain-specific mutants to identify which ankyrin repeats are crucial for specific regulatory functions

  • Use ChIP-seq to identify potential chromatin associations if ANKRD46 is implicated in transcriptional regulation

• Network analysis:

  • Apply pathway enrichment analysis to genes correlated with ANKRD46 expression

  • Use protein interaction databases to predict functional modules containing ANKRD46

  • Consider cross-species conservation of regulatory networks between human and orangutan data

What are the challenges in studying ANKRD46 from non-human primates compared to human ANKRD46?

Researching non-human primate proteins like Pongo abelii ANKRD46 presents several methodological challenges:

• Reagent availability constraints:

  • Limited commercial antibodies specifically validated for orangutan proteins

  • Fewer available cell lines and tissue samples from Pongo abelii

  • Cross-reactivity of human antibodies must be empirically validated

• Comparative experimental design considerations:

  • Need for careful primer design that accounts for sequence differences when performing PCR or qPCR

  • Importance of sequence verification when cloning from orangutan samples

  • Requirement for species-specific positive controls in expression studies

• Functional conservation assessment:

  • Design experiments to determine if the protein has conserved or divergent functions

  • Consider complementation assays where human protein is replaced with orangutan ortholog

  • Analyze evolutionary conservation patterns of specific domains and motifs

How can I design experiments to elucidate the cellular localization and trafficking of ANKRD46?

To investigate cellular localization and trafficking of ANKRD46:

• Fluorescent protein fusion strategies:

  • Generate N- and C-terminal fluorescent protein fusions (GFP, mCherry)

  • Create domain-specific deletions to identify localization signals

  • Use time-lapse imaging to track dynamic localization changes

• Immunofluorescence approaches:

  • Develop specific antibodies against Pongo abelii ANKRD46 or use cross-reactive antibodies

  • Perform co-staining with organelle markers to determine subcellular localization

  • Apply super-resolution microscopy for detailed localization studies

• Biochemical fractionation:

  • Perform subcellular fractionation followed by Western blotting

  • Compare fractionation patterns under different cellular conditions

  • Validate findings with orthogonal methods such as imaging

How does ANKRD46 in Pongo abelii relate to orangutan conservation research?

While ANKRD46 itself has not been directly linked to conservation efforts in the available literature, studying orangutan genetics and proteomics contributes to broader conservation goals:

• Population genetics applications:

  • Genetic diversity studies may include analysis of genes like ANKRD46 to assess population health

  • Comparative studies across isolated populations can provide insights into adaptive evolution

  • Current conservation efforts focus on protecting the approximately 13,000 remaining Sumatran orangutans (Pongo abelii) in the wild

• Research infrastructure:

  • Long-term monitoring stations like those in Batang Toru, Sikundur, and Suaq Balimbing provide opportunities for sample collection

  • These established research sites enable longitudinal studies of orangutan biology and ecology

  • Molecular studies can complement behavioral and ecological research at these sites

• Methodological considerations:

  • Non-invasive sampling techniques are preferable for endangered species

  • Validation of methods using captive animals before applying to wild populations

  • Ethical considerations for sample collection and research design

What bioinformatic approaches are recommended for analyzing ANKRD46 across primate species?

When conducting comparative analyses of ANKRD46 across primates:

• Sequence analysis methodology:

  • Perform multiple sequence alignment of ANKRD46 orthologs using tools like Clustal Omega or MUSCLE

  • Calculate evolutionary conservation scores for specific domains and residues

  • Identify species-specific variations in functional domains

  • Construct phylogenetic trees to visualize evolutionary relationships

• Structural bioinformatics approaches:

  • Generate homology models of ANKRD46 from different species

  • Predict functional impacts of amino acid substitutions using tools like SIFT or PolyPhen

  • Perform molecular dynamics simulations to assess potential functional differences

  • Analyze conservation of protein-protein interaction interfaces

• Expression pattern comparison:

  • Analyze available transcriptomic data to compare expression patterns across tissues

  • Identify conserved regulatory elements in promoter regions

  • Compare co-expression networks across species to identify conserved functional modules

What are common pitfalls when working with recombinant ankyrin repeat domain proteins?

Researchers should be aware of these methodological challenges:

• Protein solubility and stability issues:

  • Ankyrin repeat domains may aggregate or fold improperly when expressed recombinantly

  • Consider optimizing expression conditions (temperature, induction time)

  • Test different solubility tags (MBP, SUMO, etc.) if experiencing solubility problems

  • Verify proper folding using circular dichroism or limited proteolysis

• Functional activity assessment:

  • Develop appropriate activity assays based on predicted protein function

  • Include positive controls with known ankyrin repeat domain proteins

  • Validate that recombinant protein retains native binding properties

  • Consider the impact of tags on protein function

• Quality control requirements:

  • Perform size exclusion chromatography to ensure monodispersity

  • Use multiple orthogonal methods to confirm protein-protein interactions

  • Verify protein identity by mass spectrometry

  • Test for endotoxin contamination if using in cellular assays

How can I resolve inconsistent results in ANKRD46 expression studies?

When encountering variability in gene expression results:

• Technical considerations:

  • Evaluate RNA quality using Bioanalyzer or similar methods (RIN > 8 recommended)

  • Ensure complete DNase treatment to remove genomic DNA contamination

  • Optimize primer concentrations and annealing temperatures

  • Use multiple reference genes that have been validated for stability in your experimental system

• Experimental design improvements:

  • Increase biological replicates to account for natural variation

  • Consider time-course experiments to capture expression dynamics

  • Standardize sample collection and processing protocols

  • Control for variables like time of day, feeding status, or hormonal cycles

• Advanced troubleshooting:

  • Test for transcript variants using primers targeting different exons

  • Verify primer specificity through melt curve analysis and sequencing of PCR products

  • Consider absolute quantification methods like digital PCR for low-abundance transcripts

  • Validate expression changes at the protein level when possible