TWNK Antibody

What is the TWNK gene and what role does it play in cellular function?

The TWNK gene (also known as PEO1, Twinkle, C10orf2) encodes a hexameric DNA helicase that unwinds short stretches of double-stranded DNA in the 5' to 3' direction. This protein plays a critical role in mitochondrial DNA (mtDNA) replication by working in conjunction with mitochondrial single-stranded DNA binding protein and mtDNA polymerase gamma. The Twinkle protein localizes specifically to the mitochondrial matrix and mitochondrial nucleoids, where it participates in maintaining mitochondrial genome integrity and replication .

Research has demonstrated that mutations in the TWNK gene are associated with multiple mitochondrial disorders, including infantile onset spinocerebellar ataxia (IOSCA), progressive external ophthalmoplegia (PEO), and several mitochondrial DNA depletion syndromes . The functional importance of TWNK is highlighted in animal models, where mutations like c.409G>A (p. Ala137Thr) have been linked to conditions such as Runting and Stunting Syndrome (RSS) in chickens, characterized by mtDNA depletion .

What are the recommended applications for TWNK antibodies in research?

TWNK antibodies are primarily utilized in Western blotting (WB) applications for detecting the Twinkle protein . When selecting a TWNK antibody, researchers should consider factors such as:

• Target specificity: Confirm that the antibody specifically recognizes the Twinkle protein in your species of interest

• Application compatibility: Ensure the antibody is validated for your intended application

• Epitope location: Consider whether N-terminal, C-terminal, or internal epitope recognition is most appropriate for your experiment

Research applications of TWNK antibodies include:

• Studying mitochondrial DNA replication mechanisms

• Investigating mitochondrial depletion syndromes

• Examining pathological mutations in TWNK and their effects on protein function

• Analyzing Twinkle protein expression in different tissue types and disease states

• Monitoring mitochondrial nucleoid composition and dynamics

What is the expected molecular weight of the TWNK protein in Western blot analysis?

For chicken TWNK protein studies, researchers have successfully used rabbit anti-Twinkle antibodies (bs-11775R; Bioss, China) at a 1:1,000 dilution with goat anti-rabbit IgG-HRP secondary antibody (YJ0189; Ylesa, China) at a 1:2,500 dilution . This methodology has proven effective for detecting Twinkle protein in avian tissue samples.

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

For optimal performance and longevity of TWNK antibodies, the following storage and handling recommendations should be followed:

• Short-term storage (up to 1 week): Store at 2-8°C

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles, which can degrade antibody quality

• Stability: Typical shelf life is one year from dispatch when stored properly

When working with the antibody:

• Avoid repeated freeze-thaw cycles

• Maintain cold chain during experiments

• Follow manufacturer recommendations for dilution factors

• Consider adding preservatives like sodium azide (0.09% w/v) for solutions that will be stored

How should I optimize Western blot protocols when using TWNK antibodies?

Optimizing Western blot protocols for TWNK detection requires attention to several key parameters:

• Sample preparation:

  • Extract proteins from tissue samples using appropriate lysis buffers

  • Determine protein concentration using methods such as bicinchoninic acid assay

  • Use appropriate protease inhibitors to prevent degradation

• Gel electrophoresis:

  • 10% sodium dodecyl sulfate-polyacrylamide gels have been successfully used for TWNK protein separation

  • Load appropriate amount of protein (typically 20-50 μg per lane)

• Transfer conditions:

  • Transfer to PVDF membranes at optimal voltage/current

  • Verify transfer efficiency with reversible staining

• Blocking and antibody incubation:

  • Block membranes with appropriate blocking buffer to reduce background

  • Use primary TWNK antibody at recommended dilution (typically 1:1,000)

  • Incubate with secondary antibody (HRP-conjugated) at appropriate dilution (typically 1:2,500)

• Detection:

  • Use enhanced chemiluminescence or other detection methods according to laboratory protocols

  • Include appropriate positive and negative controls

What controls should be included when performing experiments with TWNK antibodies?

To ensure the validity and reliability of results when using TWNK antibodies, incorporate the following controls:

• Positive controls:

  • Tissue or cell lysates known to express TWNK (e.g., liver, brain, or cells with high mitochondrial content)

  • Recombinant TWNK protein where available

• Negative controls:

  • Tissues or cells with confirmed low or no TWNK expression

  • TWNK knockout or knockdown samples where available

  • Primary antibody omission control

• Loading controls:

  • Housekeeping proteins such as β-actin (e.g., mouse anti-β-actin, 3700S; CST, dilution 1:1,000)

  • Mitochondrial markers for co-localization studies

• Specificity controls:

  • Peptide competition assay to verify antibody specificity

  • Testing multiple antibodies targeting different epitopes

These controls collectively help validate experimental findings and address potential issues related to antibody cross-reactivity, sample loading variations, and technical artifacts.

How can TWNK antibodies be used to study mitochondrial DNA depletion syndromes?

TWNK antibodies serve as valuable tools for investigating mitochondrial DNA depletion syndromes (MDS) through multiple experimental approaches:

• Comparative expression analysis:

  • Quantify TWNK protein levels in affected versus normal tissues using Western blotting

  • Correlate protein expression with mtDNA copy number measurements

  • Analyze tissue-specific expression patterns in different MDS phenotypes

• Mutation impact assessment:

  • Detect mutated TWNK proteins and analyze their expression levels

  • Compare wild-type and mutant TWNK function through overexpression studies

  • Evaluate the effect of specific mutations (e.g., TWNK c.409G>A) on protein stability and function

• Protein-protein interaction studies:

  • Use co-immunoprecipitation with TWNK antibodies to identify interaction partners

  • Analyze how disease-causing mutations affect these interactions

  • Investigate the formation of the mtDNA replication complex

• Structure-function relationship analysis:

  • Study how mutations affect TWNK localization to mitochondrial nucleoids

  • Correlate structural changes with functional defects using biochemical assays

For example, research on the TWNK c.409G>A (p. Ala137Thr) mutation in chickens demonstrated that this mutation is associated with mtDNA depletion in liver tissue and correlates with growth deficiencies. Overexpression studies showed that while wild-type TWNK increases mtDNA copy number, the mutant TWNK A137T causes mtDNA depletion in vitro, providing direct evidence of the mutation's pathogenicity .

What methodological approaches can be used to investigate TWNK mutations and their effects?

Investigating TWNK mutations requires a multi-faceted approach combining molecular, cellular, and bioinformatics techniques:

• Genetic screening and sequencing:

  • PCR amplification and sequencing of TWNK genomic DNA

  • Use of primers targeting specific regions of interest (refer to Table 1 for appropriate primers)

  • Next-generation sequencing approaches for comprehensive mutation analysis

• Bioinformatics analysis of mutations:

  • Prediction of structural changes using tools like ProtParam and ProtScale

  • Analysis of physicochemical properties (molecular weight, instability index, aliphatic index, GRAVY score)

  • Secondary structure modeling using tools like SOPMA to assess changes in alpha helices, extended strands, beta turns, and random coils

• Recombinant protein expression:

  • Cloning of wild-type and mutant TWNK into expression vectors

  • Site-directed mutagenesis to generate specific mutations

  • Expression in appropriate cell systems for functional studies

• Functional assessments:

  • mtDNA copy number analysis by qPCR

  • Helicase activity assays to measure enzymatic function

  • Cell growth and viability studies to assess phenotypic impact

In a study examining the TWNK c.409G>A mutation, researchers observed that the mutation altered several physiological and biochemical properties, including reduced molecular weight, instability index, aliphatic index, and GRAVY score. Additionally, local hydrophobicity at and near the mutated residue was reduced, and the content of alpha helices was diminished compared to the wild-type protein .

How can I use TWNK antibodies to study protein-protein interactions in mitochondrial nucleoids?

Studying TWNK protein interactions within mitochondrial nucleoids requires specialized techniques:

• Co-immunoprecipitation (Co-IP):

  • Use TWNK antibodies to pull down the protein complex

  • Identify interaction partners through Western blotting or mass spectrometry

  • Compare interaction profiles between wild-type and mutant TWNK

• Proximity ligation assay (PLA):

  • Detect and visualize protein interactions in situ

  • Combine TWNK antibodies with antibodies against potential interaction partners

  • Quantify interaction signals in different cellular conditions

• Immunofluorescence microscopy:

  • Visualize co-localization of TWNK with other nucleoid proteins

  • Analyze dynamics of protein interactions during mtDNA replication

  • Assess the impact of mutations on localization patterns

• FRET/BRET assays:

  • Measure direct protein interactions through fluorescence/bioluminescence resonance energy transfer

  • Tag TWNK and interaction partners with appropriate fluorophores

  • Evaluate interaction dynamics in living cells

• Chromatin immunoprecipitation (ChIP):

  • Use TWNK antibodies to identify DNA-binding sites

  • Map interaction with mtDNA replication origins

  • Compare binding profiles between wild-type and mutant proteins

When planning these experiments, researchers should consider using affinity-purified antibodies with minimal cross-reactivity to ensure specific detection of TWNK interactions .

What are common issues when working with TWNK antibodies and how can they be resolved?

Researchers may encounter several challenges when working with TWNK antibodies. Here are common issues and their solutions:

For optimal Western blot results with TWNK antibodies, researchers have successfully used 10% SDS-PAGE gels, PVDF membranes, and detection with enhanced chemiluminescence systems . Additionally, ensuring proper sample preparation with protease inhibitors and determining protein concentration using bicinchoninic acid assay before loading can significantly improve results .

How can I validate the specificity of my TWNK antibody for my target species?

Validating antibody specificity is crucial for reliable results, especially when working with different species. Follow these approaches:

• Sequence homology analysis:

  • Compare the immunogen sequence with the corresponding sequence in your target species

  • Assess the degree of conservation at the antibody recognition site

  • Higher homology increases the likelihood of cross-reactivity

• Positive and negative controls:

  • Use tissues/cells known to express or lack TWNK in your species

  • Compare with recombinant proteins where available

  • Include samples from knockout/knockdown models if accessible

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide

  • A specific signal should be significantly reduced or eliminated

  • Non-specific signals will likely remain unchanged

• Multiple antibody comparison:

  • Test antibodies targeting different epitopes of TWNK

  • Consistent results across antibodies support specificity

  • Discrepancies may indicate non-specific binding

• Western blot analysis:

  • Verify the detection of a band at the expected molecular weight

  • For example, human TWNK is predicted to be approximately 75 kDa

  • Species variations in size should be considered based on sequence information

When selecting a TWNK antibody for cross-species applications, carefully review the manufacturer's specifications regarding expected reactivity. Some antibodies, like the polyclonal rabbit anti-Twinkle (bs-11775R; Bioss, China), have been successfully used in multiple species including chicken .

What considerations should be made when designing experiments to study TWNK mutations?

When designing experiments to investigate TWNK mutations, consider these methodological factors:

• Mutation selection and characterization:

  • Prioritize clinically relevant mutations with known pathogenicity

  • Consider the domain location of mutations (e.g., primase domain, helicase domain)

  • Use bioinformatics tools to predict structural and functional impacts

• Expression system selection:

  • Choose appropriate vectors (e.g., pcDNA3.1) for mammalian expression

  • Consider using inducible systems for toxic mutations

  • Include appropriate selection markers for stable expression

• Cloning strategy:

  • Use site-directed mutagenesis to introduce specific mutations

  • Verify mutations by sequencing before functional studies

  • Consider tag addition for detection if antibodies are limiting

• Functional readouts:

  • Measure mtDNA copy number changes as a primary readout

  • Assess cell growth and viability as secondary phenotypes

  • Evaluate mitochondrial function (respiration, membrane potential)

• Control selection:

  • Include wild-type TWNK as positive control

  • Use empty vector transfection as negative control

  • Consider including known pathogenic mutations as reference points

For example, researchers studying the TWNK c.409G>A mutation generated wild-type and mutant expression constructs using a cloning strategy involving PCR amplification of the TWNK coding sequence, followed by cloning into the pcDNA3.1 vector through pMD18-T cloning vector using the EcoRI and HindIII restriction sites . This approach allowed direct comparison of wild-type and mutant TWNK effects on mtDNA replication.

How are TWNK antibodies used in the study of mitochondrial diseases?

TWNK antibodies serve as essential tools in mitochondrial disease research through several applications:

• Diagnostic marker assessment:

  • Evaluate TWNK protein levels in patient samples

  • Compare expression patterns between disease and control tissues

  • Correlate protein abundance with disease severity

• Pathogenic mechanism investigation:

  • Analyze how mutations affect protein stability and expression

  • Study subcellular localization changes in disease states

  • Examine interactions with other replication factors

• Animal model validation:

  • Confirm the presence of TWNK mutations in disease models

  • Assess protein expression in affected tissues

  • Correlate biochemical findings with phenotypic manifestations

• Therapeutic development support:

  • Monitor TWNK protein levels during experimental treatments

  • Assess restoration of protein function in gene therapy approaches

  • Evaluate compound effects on TWNK stability or activity

In chicken models of Runting and Stunting Syndrome (RSS), TWNK antibodies were instrumental in confirming that the TWNK c.409G>A mutation affects protein function, leading to mtDNA depletion. These findings established a direct molecular link between the mutation and the observed phenotype, characterized by reduced body weight, poor performance, and growth deficiencies .

What techniques can be used to assess the impact of TWNK mutations on mtDNA maintenance?

Assessing how TWNK mutations affect mtDNA maintenance requires multiple complementary techniques:

• mtDNA copy number analysis:

  • Quantitative PCR comparing mitochondrial to nuclear DNA ratios

  • Digital droplet PCR for absolute quantification

  • Southern blotting for analyzing mtDNA depletion patterns

• mtDNA integrity assessment:

  • Long-range PCR to detect large-scale deletions

  • Next-generation sequencing to identify point mutations

  • Single-molecule analysis to examine heteroplasmy levels

• Protein function studies:

  • In vitro helicase activity assays with purified proteins

  • DNA binding assays to assess substrate recognition

  • Overexpression studies comparing wild-type and mutant effects

• Cellular phenotype characterization:

  • Mitochondrial membrane potential measurements

  • Respiratory chain complex activity assays

  • ATP production and oxygen consumption analysis

Research has demonstrated that overexpression of wild-type TWNK increases mtDNA copy number, whereas overexpression of mutant variants like TWNK A137T causes mtDNA depletion in vitro . This experimental approach provides direct evidence of the functional consequences of TWNK mutations on mtDNA maintenance and can be applied to study various disease-associated mutations.

How do we correlate TWNK mutations with phenotypic manifestations in animal models?

Establishing correlations between TWNK mutations and phenotypic outcomes in animal models involves these methodological approaches:

• Genotype-phenotype correlation studies:

  • Compare multiple animals with the same mutation

  • Assess dose-dependent effects in heterozygous vs. homozygous models

  • Track phenotypic progression over time

• Tissue-specific analysis:

  • Examine TWNK expression and mtDNA content across tissues

  • Correlate tissue-specific mtDNA depletion with organ dysfunction

  • Identify particularly vulnerable tissues

• Biochemical parameter assessment:

  • Measure growth parameters, body weight, and organ weights

  • Analyze tissue-specific energy metabolism

  • Evaluate mitochondrial function in affected tissues

• Statistical association analysis:

  • Perform association studies between mutation and phenotypic traits

  • Use appropriate statistical methods to establish significance

  • Control for confounding factors

In a study of SLD chickens with the TWNK c.409G>A mutation, researchers found significant associations between this mutation and economic traits including body weight, daily gain, pectoralis weight, crureus weight, and abdominal fat weight . The study utilized 339 normal SLD chickens to establish these associations, demonstrating the importance of adequate sample size in correlation studies.