SUPV3L1 Antibody

What is SUPV3L1 and what cellular processes does it participate in?

SUPV3L1 (suppressor of var1, 3-like 1) is an ATP-dependent RNA helicase that forms a critical component of the mitochondrial degradosome complex. It plays an essential role in mitochondrial RNA metabolism by unwinding secondary structures of mitochondrial RNA (mtRNA) and facilitating the degradation of mtRNA molecules . This protein is integral to the degradation of non-coding mitochondrial transcripts (MT-ncRNA) and tRNA-like molecules . It's involved in mitochondrial RNA surveillance and degradation, processing primary transcripts into mature mRNAs and rRNAs, and preventing the accumulation of mitochondrial double-stranded RNA (mtdsRNA) . Dysfunction of SUPV3L1 can lead to severe mitochondrial disorders with diverse clinical presentations.

What are the recommended applications and dilutions for SUPV3L1 antibody?

Below is a comprehensive table of recommended applications and dilutions for SUPV3L1 antibody (specifically for antibody 12826-1-AP):

It is important to note that these dilutions should be optimized for each specific experimental system to obtain optimal results . For IHC applications, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may be used as an alternative .

What is the expected molecular weight when detecting SUPV3L1?

When working with SUPV3L1 antibodies, researchers should be aware of potential discrepancies between calculated and observed molecular weights:

• Calculated molecular weight: 88 kDa (786 amino acids)

• Observed molecular weight in experimental systems: 70-80 kDa

This difference between calculated and observed molecular weights may result from post-translational modifications, protein processing, or degradation. When performing Western blot analysis, expect to observe bands in the 70-80 kDa range .

How should SUPV3L1 antibody be stored for optimal performance?

For optimal antibody performance and longevity:

• Store at -20°C in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• The antibody is stable for one year after shipment when properly stored

• Aliquoting is unnecessary for -20°C storage

• Small volume preparations (20 μl) contain 0.1% BSA as a stabilizer

Following these storage recommendations will help maintain antibody activity and specificity throughout your research project.

How can I validate SUPV3L1 antibody specificity in my experimental system?

Validating antibody specificity is crucial for obtaining reliable research results. For SUPV3L1 antibody validation, consider the following approach:

• Positive controls: Use cell lines known to express SUPV3L1, such as HEK-293, PC-3, or HeLa cells

• Knockout/knockdown validation: Compare antibody reactivity between wild-type and SUPV3L1-depleted samples to confirm specificity

• Multiple detection methods: Confirm findings using different techniques (e.g., WB, IF, IHC) to strengthen confidence in antibody specificity

• Immunoprecipitation followed by mass spectrometry: Perform IP using the SUPV3L1 antibody followed by mass spectrometry analysis to confirm target capture

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide to demonstrate signal reduction in the presence of the competing peptide

Published literature showing successful application of the antibody can provide additional validation support. The antibody 12826-1-AP has been cited in at least two publications for Western blot applications .

What are the optimal protocols for studying SUPV3L1's role in the mitochondrial degradosome complex?

To investigate SUPV3L1's role in the mitochondrial degradosome complex, consider these methodological approaches:

• Co-immunoprecipitation studies: Use SUPV3L1 antibody to pull down associated proteins, particularly PNPT1, which forms the mitochondrial RNA degradosome with SUPV3L1 . Follow with Western blot or mass spectrometry analysis.

• Mitochondrial isolation and fractionation:

  • Isolate intact mitochondria using differential centrifugation

  • Perform subfractionation to separate mitochondrial compartments

  • Confirm SUPV3L1 localization using the antibody in Western blot analysis

• RNA-protein interaction studies:

  • RNA immunoprecipitation (RIP) to identify bound mitochondrial RNAs

  • Cross-linking immunoprecipitation (CLIP) for more precise mapping of RNA-protein interactions

• Functional assays:

  • Measure mtRNA degradation rates in cells with normal vs. depleted SUPV3L1

  • Assess the impact on mitochondrial function using respirometry

  • Evaluate mitochondrial translation using techniques like mePROD mt (mitochondrial multiplexed enhanced protein dynamic)

• Protein complex analysis:

  • Blue native PAGE to analyze intact complexes

  • Size exclusion chromatography coupled with Western blot detection

These methodological approaches will help elucidate SUPV3L1's specific functions within the mitochondrial degradosome complex and its interactions with other components of the mitochondrial RNA degradation machinery.

How do SUPV3L1 mutations affect mitochondrial function and RNA processing?

Pathogenic variants in SUPV3L1 have significant impacts on mitochondrial function and RNA processing. Based on clinical and molecular studies:

• Mitochondrial RNA accumulation: Mutations in SUPV3L1 lead to the accumulation of mitochondrial double-stranded RNA (mtdsRNA), which would normally be degraded by the mitochondrial degradosome complex .

• Immune response activation: The accumulated mtdsRNA can dysregulate interferon signaling and potentially activate antiviral immune responses, contributing to disease pathology .

• Oxidative phosphorylation defects: ELISA analysis of patient fibroblasts with SUPV3L1 mutations has shown a 2-fold increase in Complex I content, potentially representing a compensatory response to mitochondrial dysfunction .

• Clinical manifestations: SUPV3L1 mutations lead to a spectrum of phenotypes including:

  • Neurological symptoms (ataxia, spastic paraparesis, cognitive deficit)

  • Optic atrophy

  • Horizontal gaze-evoked nystagmus

  • Hypopigmented skin patches

• Mouse model findings: Studies in mice with Supv3L1 knockout have demonstrated:

  • Embryonic lethality with complete deletion

  • Premature aging phenotypes with conditional knockout

  • Skin abnormalities resembling those in human patients

  • Sarcopenia and loss of adipose tissue

What experimental approaches can help elucidate SUPV3L1 interactions with the RNA processing machinery?

To investigate SUPV3L1's interactions with other components of the RNA processing machinery, consider these advanced experimental approaches:

• Proximity-dependent biotinylation (BioID or TurboID):

  • Generate SUPV3L1-BioID fusion proteins

  • Identify proximal proteins through streptavidin pulldown followed by mass spectrometry

  • This approach can reveal both stable and transient interactions in the native cellular environment

• Quantitative proteomic analysis:

  • Compare protein abundance in wild-type versus SUPV3L1-depleted cells

  • Apply techniques like mePROD (multiplexed enhanced protein dynamic) to monitor newly synthesized proteins

  • Use mePROD mt for more selective quantification of proteins imported into mitochondria

• Mitochondrial transcriptome analysis:

  • Perform RNA-Seq specifically on mitochondrial RNA

  • Analyze changes in mitochondrial transcript processing and abundance in SUPV3L1-mutant cells

  • Investigate the accumulation of non-coding RNAs and aberrant transcripts

• Structural studies:

  • Cryo-EM or X-ray crystallography of SUPV3L1 alone or in complex with RNA

  • Molecular dynamics simulations to understand conformational changes during RNA binding and unwinding

• In vitro reconstitution assays:

  • Purify recombinant SUPV3L1 and associated factors

  • Assess RNA helicase activity using defined RNA substrates

  • Reconstitute the mitochondrial degradosome complex in vitro to study its mechanistic properties

These approaches will provide comprehensive insights into SUPV3L1's functional interactions within the mitochondrial RNA processing machinery.

How can SUPV3L1 antibodies be used in the diagnosis of SUPV3L1-associated disorders?

While genetic testing remains the gold standard for diagnosing SUPV3L1-associated disorders, antibody-based approaches can be valuable complementary tools:

• Tissue expression analysis: SUPV3L1 antibodies can be used for immunohistochemistry (IHC) to examine protein expression in patient tissue samples. The recommended dilution for IHC applications is 1:250-1:1000 .

• Protein quantification in patient samples:

  • Western blot analysis of fibroblasts or muscle biopsies to assess SUPV3L1 protein levels

  • Compare with control samples to identify potential differences in expression or molecular weight

• Functional assays in patient-derived cells:

  • Immunofluorescence microscopy to assess SUPV3L1 subcellular localization

  • Co-localization studies with mitochondrial markers

  • Evaluation of mitochondrial morphology and distribution

• Biomarker development:

  • Investigate whether SUPV3L1 or its downstream targets could serve as biomarkers for disease progression

  • Correlate protein levels with clinical severity

It's important to note that SUPV3L1-associated disorders are rare, with only a small number of documented cases . The presentation includes a variable spectrum of symptoms, making comprehensive diagnostic approaches necessary.

What are the phenotypic characteristics of SUPV3L1-associated mitochondrial disease?

SUPV3L1-associated mitochondrial disease presents with a diverse array of symptoms. Based on documented cases, the clinical manifestations include:

The disease typically has an early onset, with symptoms appearing before 12 months of age in most cases, though the patient described in the recent case report exhibited symptoms at 1 year and 8 months . The clinical course is generally mild and progressive, with variable severity across patients. Some patients may exhibit a less severe form, maintaining the ability to walk independently with occasional support .

How can I optimize Western blot protocols for SUPV3L1 detection?

For optimal detection of SUPV3L1 in Western blot applications, consider the following recommendations:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • For mitochondrial proteins, consider mitochondrial isolation before lysis

  • Avoid excessive freeze-thaw cycles of samples

• Protein loading and transfer:

  • Load 20-50 μg of total protein lysate

  • Use positive control lysates from HEK-293, PC-3, or HeLa cells

  • Consider using wet transfer for large proteins

• Antibody dilution and incubation:

  • Start with a 1:5000 dilution of SUPV3L1 antibody (12826-1-AP) for Western blot

  • Optimize by testing a range from 1:2000 to 1:10000

  • Incubate overnight at 4°C for primary antibody

• Detection considerations:

  • Look for bands in the 70-80 kDa range (observed molecular weight)

  • Be aware that the calculated molecular weight (88 kDa) differs from observed weight

  • Use appropriate molecular weight markers spanning 50-100 kDa

• Troubleshooting weak signals:

  • Increase antibody concentration

  • Extend incubation time

  • Consider using signal enhancement systems

  • Ensure fresh transfer buffers and blocking solutions

Following these optimization steps will help ensure specific and robust detection of SUPV3L1 in your experimental system.

What are the key considerations for immunoprecipitation studies with SUPV3L1 antibody?

For successful immunoprecipitation of SUPV3L1 and its interacting partners:

• Antibody amount and lysate ratio:

  • Use 0.5-4.0 μg of SUPV3L1 antibody for 1.0-3.0 mg of total protein lysate

  • Adjust based on expression level in your specific cell type

• Lysate preparation:

  • Use gentle lysis conditions to preserve protein-protein interactions

  • Consider crosslinking for transient interactions

  • Include appropriate protease and phosphatase inhibitors

• Immunoprecipitation protocol:

  • Pre-clear lysate with protein A/G beads to reduce background

  • Incubate antibody with lysate overnight at 4°C

  • Use gentle washing conditions to preserve interactions

• Controls and validation:

  • Include IgG control to identify non-specific binding

  • Verify successful immunoprecipitation by Western blot

  • Consider HeLa cells as a positive control system

• Co-immunoprecipitation considerations:

  • For identifying SUPV3L1 interaction partners, such as PNPT1 in the mitochondrial degradosome

  • Analyze by both Western blot and mass spectrometry

  • Validate findings with reciprocal immunoprecipitation

These methodological considerations will help ensure specific and efficient immunoprecipitation of SUPV3L1 and its interacting partners.

What are emerging research areas involving SUPV3L1 in mitochondrial biology?

Several promising research directions are emerging in the field of SUPV3L1 and mitochondrial biology:

• Role in immune signaling: Further investigation into how SUPV3L1 dysfunction leads to mitochondrial double-stranded RNA accumulation and subsequent immune response activation . This research area connects mitochondrial RNA processing defects with innate immune pathways.

• Therapeutic potential: Exploring targeted therapeutic interventions for SUPV3L1-associated disorders. Understanding the molecular mechanisms underlying the pathology could lead to novel treatment approaches .

• Interaction with nuclear-encoded transcripts: Investigating the relationship between cytosolic N6AMT1-dependent translation and mitochondrial function, particularly how it affects RNA processing factors like SUPV3L1 .

• Role in aging and age-related diseases: Based on mouse models showing premature aging phenotypes with Supv3L1 knockout, exploring the connection between SUPV3L1 function and aging processes .

• Tissue-specific functions: Investigating why SUPV3L1 dysfunction affects certain tissues more severely than others, particularly focusing on neurological and dermatological manifestations .

These emerging research areas represent important frontiers in understanding SUPV3L1's role in mitochondrial biology and potential therapeutic approaches for related disorders.

How can advanced techniques like Cryo-EM and AlphaFold contribute to SUPV3L1 research?

Advanced structural biology techniques offer powerful approaches to understanding SUPV3L1 function:

• Cryo-electron microscopy (Cryo-EM):

  • Determine high-resolution structures of SUPV3L1 alone or in complex with RNA substrates

  • Visualize conformational changes during ATP binding and hydrolysis

  • Elucidate the structure of the entire mitochondrial degradosome complex

  • Identify potential binding sites for small molecule modulators

• AlphaFold and other AI-based structure prediction:

  • Generate predicted structures of SUPV3L1 and its complexes

  • Model the impact of disease-associated mutations on protein structure

  • Predict protein-protein interaction interfaces

  • Guide experimental design for mutagenesis studies

• Integration of structural data with functional studies:

  • Correlate structural features with biochemical activities

  • Design structure-based mutations to test mechanistic hypotheses

  • Develop small molecule modulators of SUPV3L1 activity

• Single-molecule approaches:

  • Visualize SUPV3L1 helicase activity in real-time

  • Measure kinetic parameters of RNA unwinding

  • Observe conformational dynamics during catalytic cycles

These advanced techniques will provide unprecedented insights into the molecular mechanisms of SUPV3L1 function and how mutations lead to disease, potentially opening new avenues for therapeutic intervention.