SUPV3L1 Antibody, FITC conjugated

What is SUPV3L1 and why is it an important research target?

SUPV3L1 (Suppressor of var1, 3-like 1) is a major helicase player in mitochondrial RNA metabolism and a key component of the mitochondrial degradosome (mtEXO) complex. This protein degrades 3' overhang double-stranded RNA with 3'-to-5' directionality in an ATP-dependent manner . SUPV3L1 is crucial for:

• Degradation of non-coding mitochondrial transcripts (MT-ncRNA) and tRNA-like molecules

• RNA surveillance in mitochondria, regulating stability of mature mRNAs

• Removal of aberrantly formed mRNAs and rapid degradation of non-coding processing intermediates

• Maintaining mitochondrial DNA integrity

Recent research has linked SUPV3L1 mutations to mitochondrial disorders with neurological manifestations, making it an important target for understanding mitochondrial dysfunction and related diseases .

What are the optimal storage conditions for FITC-conjugated SUPV3L1 antibodies?

FITC-conjugated SUPV3L1 antibodies should be stored at -20°C, where they remain stable for approximately one year after shipment . For long-term stability:

• Aliquot the antibody to avoid repeated freeze/thaw cycles

• Keep protected from light to prevent photobleaching of the FITC fluorophore

• Store in the buffer provided by the manufacturer (typically PBS with 0.02% sodium azide and 50% glycerol pH 7.3)

• For 20μL sized preparations, note that they may contain 0.1% BSA as a stabilizer

Which cell lines have been validated for SUPV3L1 detection with antibodies?

The following cell lines have been experimentally validated for SUPV3L1 detection:

For immunofluorescence studies specifically with FITC-conjugated antibodies, HeLa cells are consistently reported as suitable models for SUPV3L1 localization studies .

What are the recommended dilutions for FITC-conjugated SUPV3L1 antibodies in different applications?

Optimal dilutions vary by application and specific antibody preparation:

Always perform an antibody titration experiment in your specific model system to determine optimal working dilution .

How do I troubleshoot discrepancies between observed and calculated molecular weights for SUPV3L1?

Discrepancies between calculated (88 kDa) and observed (70-80 kDa) molecular weights for SUPV3L1 are common and can be attributed to several factors :

• Post-translational modifications: SUPV3L1 may undergo phosphorylation or other modifications that alter mobility

• Protein processing: The mitochondrial targeting sequence may be cleaved upon import into mitochondria

• Protein conformation: The highly structured nature of helicases can affect SDS-PAGE migration

• Splice variants: Different isoforms may be detected in different tissues

Troubleshooting approaches:

• Use positive control lysates from validated cell lines (HeLa, HEK-293)

• Perform subcellular fractionation to confirm mitochondrial localization

• Include phosphatase treatment of lysates to identify potential phosphorylation

• Compare results with antibodies targeting different epitopes of SUPV3L1

What is the significance of the C-terminal region (aa 739-786) of SUPV3L1 for antibody selection and experimental design?

The C-terminal region of SUPV3L1 (amino acids 739-786) has specific biological importance that researchers should consider when selecting antibodies :

• Functional domains: This region contains:

  • A predicted conserved double amphipathic helix

  • A potential RNA interaction domain (aa 737-777)

  • A potential nuclear localization signal (RRKK stretch)

• Experimental considerations:

  • Antibodies targeting this region (such as those using immunogens aa 700-C-terminus) may be useful for studying SUPV3L1 nuclear-mitochondrial shuttling

  • C-terminal truncation mutants (as observed in patients with SUPV3L1-associated diseases) show altered protein function while maintaining stability

  • FITC-conjugated antibodies against the C-terminal region can be particularly valuable for co-localization studies to determine subcellular distribution patterns

For dual localization studies, combining a FITC-conjugated C-terminal targeting antibody with antibodies against known mitochondrial markers can provide insights into SUPV3L1 trafficking between compartments .

How can FITC-conjugated SUPV3L1 antibodies be optimized for studying mitochondrial RNA degradosome complexes?

To optimize FITC-conjugated SUPV3L1 antibodies for studying mitochondrial RNA degradosome complexes:

• Sample preparation:

  • Use mitochondrial isolation protocols that preserve protein-protein interactions

  • Consider mild fixation methods that maintain complex integrity while allowing antibody access

  • For live-cell imaging, mitochondrial membrane potential dyes can be used alongside if spectral overlap is managed

• Co-immunoprecipitation optimization:

  • When using FITC-conjugated antibodies for IP followed by fluorescence detection, use anti-FITC secondary antibodies for pull-down

  • Validate interactions with known degradosome partners like PNPase (polynucleotide phosphorylase)

• Advanced microscopy approaches:

  • Super-resolution microscopy can resolve degradosome complexes within mitochondrial subcompartments

  • FRET (Fluorescence Resonance Energy Transfer) assays using FITC-conjugated SUPV3L1 antibodies and rhodamine-conjugated partner protein antibodies can confirm direct interactions

• Controls and validation:

  • Include SUPV3L1 knockdown cells as negative controls

  • RNA degradation assays should be performed to correlate localization with function

  • Compare subcellular distributions in different metabolic states

What considerations are important when analyzing SUPV3L1 in patient-derived samples with mitochondrial disease?

When analyzing SUPV3L1 in patient-derived samples with mitochondrial disease:

• Clinical sample handling:

  • Process samples rapidly to preserve mitochondrial integrity

  • Consider fixation methods that preserve both protein epitopes and mitochondrial morphology

  • Standardize sample collection timing due to potential circadian variation in mitochondrial function

• Mutation-specific considerations:

  • Recent studies have identified SUPV3L1 mutations in patients with varied neurological presentations

  • Patients with C-terminal truncations show unique patterns of mitochondrial RNA accumulation

  • Compound heterozygous variants (e.g., c.272-2A>G and c.1924A>C; p.Ser642Arg) show splicing changes

• Analytical approaches:

  • Compare SUPV3L1 distribution patterns between patient and control samples

  • Analyze mitochondrial RNA species accumulation using RNA-FISH together with FITC-SUPV3L1 antibody staining

  • Assess mitochondrial complex assembly (particularly Complex I, which shows 2-fold increase in patient fibroblasts)

  • Correlate findings with clinical phenotypes (ataxia, spastic paraparesis, cognitive deficit, optic atrophy, skin hypopigmentation)

• Experimental controls:

  • Age-matched controls are essential due to age-related changes in mitochondrial function

  • Patient-derived cell lines should be passage-matched with controls

  • Consider tissue-specific differences when analyzing SUPV3L1 expression patterns

What fixation and permeabilization protocols are optimal for FITC-conjugated SUPV3L1 antibodies in immunofluorescence?

For optimal immunofluorescence results with FITC-conjugated SUPV3L1 antibodies:

• Fixation protocols:

  • 4% paraformaldehyde (10-15 minutes at room temperature) preserves mitochondrial morphology

  • Avoid methanol fixation which can extract mitochondrial lipids and affect organelle integrity

  • For co-localization studies with mitochondrial DNA, consider using a combination of paraformaldehyde and glutaraldehyde (0.1-0.5%)

• Permeabilization optimization:

  • Digitonin (25-50 μg/mL) provides selective permeabilization of the plasma membrane while preserving mitochondrial membranes

  • For full access to mitochondrial matrix, use 0.1-0.2% Triton X-100 after fixation

  • Titrate detergent concentration carefully as over-permeabilization can disrupt mitochondrial ultrastructure

• Blocking considerations:

  • Use 5% BSA or 10% normal serum from the same species as the secondary antibody

  • Include 0.1% Tween-20 in blocking buffer to reduce background

  • For highly specific detection, consider using a combination of BSA and cold fish gelatin

• Mitochondrial counterstaining:

  • Use MitoTracker dyes before fixation or antibodies against mitochondrial markers (TOMM20, COX IV) that don't spectrally overlap with FITC

  • When selecting counterstains, consider that SUPV3L1 may also have nuclear localization under certain conditions

How can multiple epitope targeting be utilized to validate SUPV3L1 antibody specificity?

Multiple epitope targeting is crucial for validating SUPV3L1 antibody specificity:

• Comparative analysis of different domain-targeting antibodies:

  • N-terminal antibodies (e.g., AA 93-121)

  • Middle region antibodies (e.g., AA 499-786)

  • C-terminal antibodies (e.g., AA 700-C-terminus)

• Validation approaches:

  • siRNA/shRNA knockdown of SUPV3L1 should reduce signal with all antibodies

  • Overexpression of tagged SUPV3L1 should show co-localization with antibody staining

  • For FITC-conjugated antibodies specifically, pre-absorption with immunizing peptide should abolish fluorescence

  • Comparison of staining patterns across multiple cell types with known SUPV3L1 expression levels

• Domain-specific considerations:

  • The C-terminal region contains functional domains that may be masked in certain protein-protein interactions

  • N-terminal antibodies may not detect processed forms after mitochondrial import

  • Phosphorylation-sensitive epitopes may show variable detection depending on cellular metabolic status

• Technical controls:

  • Include isotype control antibodies conjugated to FITC at the same concentration

  • Secondary-only controls when using indirect immunofluorescence methods

  • When comparing multiple antibodies, standardize imaging parameters across samples

What are the optimal protocols for dual-color imaging using FITC-conjugated SUPV3L1 antibodies and mitochondrial markers?

For effective dual-color imaging:

• Compatible fluorophore selection:

  • Pair FITC (excitation ~495nm, emission ~520nm) with fluorophores having minimal spectral overlap

  • Recommended combinations:

    • FITC-SUPV3L1 + MitoTracker Deep Red (excitation ~644nm, emission ~665nm)

    • FITC-SUPV3L1 + Alexa Fluor 647-conjugated TOMM20 antibody

    • FITC-SUPV3L1 + DAPI for nuclear counterstaining (to assess potential nuclear localization)

• Sequential imaging approach:

  • Acquire FITC channel first to minimize photobleaching

  • Use narrow bandpass filters to minimize bleed-through

  • Consider linear unmixing algorithms for closely overlapping signals

• Sample preparation optimization:

  • For optimal mitochondrial morphology preservation, image cells grown on glass-bottom dishes

  • Consider super-resolution techniques (STED, SIM, PALM) for resolving SUPV3L1 distribution within mitochondrial subcompartments

  • For tissue sections, use thin sections (5-8 μm) and optimize antigen retrieval (TE buffer pH 9.0 recommended)

• Colocalization analysis:

  • Perform quantitative colocalization analysis using Pearson's correlation coefficient

  • Assess mitochondrial morphology and SUPV3L1 distribution patterns under different cellular states (e.g., oxidative stress, mtDNA depletion)

  • Compare distribution in control versus patient-derived cells with known SUPV3L1 mutations

How should researchers interpret differences in SUPV3L1 localization between cell types and disease models?

Interpretation of SUPV3L1 localization differences:

• Cell type-specific considerations:

  • SUPV3L1 shows widespread expression but with tissue-specific intensity differences

  • Highly metabolic tissues (brain, heart, liver) may show stronger mitochondrial localization

  • Proliferating cells show different mitochondrial dynamics than post-mitotic cells, affecting SUPV3L1 distribution

  • Compare localization patterns across multiple validated cell lines (HeLa, HEK-293, NIH/3T3)

• Disease model analysis:

  • In mitochondrial disease models, assess correlation between SUPV3L1 localization and:

    • Mitochondrial RNA processing defects

    • Mitochondrial membrane potential changes

    • mtDNA copy number and integrity

    • Respiratory chain complex assembly (particularly Complex I)

  • Analyze temporal changes in localization during disease progression

• Functional correlation approaches:

  • Combine imaging with functional assays (oxygen consumption, ATP production)

  • Assess mitochondrial RNA processing efficiency in regions with high versus low SUPV3L1 concentration

  • Correlate SUPV3L1 distribution with markers of mitochondrial stress or dysfunction

• Quantitative analysis frameworks:

  • Develop standardized quantification methods for SUPV3L1 mitochondrial/nuclear distribution ratios

  • Compare SUPV3L1 colocalization with different mitochondrial sub-compartment markers

  • Assess correlation between SUPV3L1 distribution patterns and clinical manifestations in patient samples

How can FITC-conjugated SUPV3L1 antibodies facilitate the study of mitochondrial RNA granules?

FITC-conjugated SUPV3L1 antibodies offer unique advantages for studying mitochondrial RNA granules:

• Live-cell imaging applications:

  • While antibodies typically require cell fixation, cell-permeable FITC-conjugated antibody fragments could enable tracking of SUPV3L1-containing complexes in living cells

  • Combined with RNA-binding dyes, these could reveal dynamics of RNA processing bodies

• High-resolution localization analysis:

  • Super-resolution microscopy using FITC-SUPV3L1 antibodies can map precise locations of RNA processing machinery within mitochondrial subdomains

  • Correlative light and electron microscopy (CLEM) can bridge fluorescence detection with ultrastructural analysis

• Functional RNA granule studies:

  • FITC-SUPV3L1 antibodies can identify sites of active RNA processing when combined with nascent RNA labeling techniques

  • Co-localization with other degradosome components can reveal assembly/disassembly dynamics under different cellular conditions

  • RNA species-specific probes used alongside SUPV3L1 antibodies can identify substrate preferences in different mitochondrial subcompartments

• Disease-relevant applications:

  • Compare RNA granule formation in cells with wild-type versus mutant SUPV3L1

  • Assess changes in granule dynamics during mitochondrial stress responses

  • Investigate the relationship between SUPV3L1-containing granules and mtRNA accumulation in patient cells

What emerging technologies can be combined with FITC-SUPV3L1 antibodies to advance mitochondrial research?

Emerging technologies that complement FITC-SUPV3L1 antibody applications:

• Proximity labeling approaches:

  • APEX2-SUPV3L1 fusion proteins can be used alongside FITC-antibody detection to identify transient interaction partners

  • BioID or TurboID-based proximity labeling can map the SUPV3L1 interactome in different cellular compartments

• Single-molecule tracking:

  • Combining sparse labeling techniques with FITC-conjugated Fab fragments against SUPV3L1 could enable single-molecule tracking

  • This approach could reveal the dynamics of individual SUPV3L1 molecules between different mitochondrial microdomains

• Cryo-electron tomography integration:

  • Correlative light and electron microscopy using FITC-SUPV3L1 antibodies as fiducial markers

  • This combination would bridge molecular identification with structural context of mitochondrial RNA processing complexes

• CRISPR-based approaches:

  • CRISPR activation or interference systems targeting SUPV3L1 combined with FITC-antibody detection to assess dosage effects

  • Gene editing to introduce fluorescent tags at the endogenous SUPV3L1 locus, with antibody validation

  • CRISPR-based mitochondrial DNA editing to study SUPV3L1 responses to mtDNA alterations

How can researchers distinguish between mitochondrial and nuclear pools of SUPV3L1 using immunofluorescence approaches?

To differentiate mitochondrial from nuclear SUPV3L1 pools:

• Subcellular fractionation validation:

  • Perform western blots on purified mitochondrial, nuclear, and cytosolic fractions

  • Use FITC-conjugated SUPV3L1 antibodies in immunofluorescence to compare with fractionation results

  • Include appropriate compartment markers (TOMM20 for mitochondria, Lamin B1 for nuclear envelope)

• Advanced microscopy approaches:

  • Z-stack confocal imaging with deconvolution to precisely localize signals in 3D

  • Airyscan or structured illumination microscopy for improved resolution of mitochondrial and nuclear signals

  • Spectral unmixing to distinguish true FITC signal from potential autofluorescence

• Selective permeabilization:

  • Digitonin at low concentrations (25 μg/mL) selectively permeabilizes plasma membrane while leaving nuclear envelope intact

  • This approach allows antibody access to cytoplasmic and mitochondrial pools while excluding nuclear pools

  • Sequential permeabilization with increasing detergent concentrations can reveal different subcellular pools

• Functional validation approaches:

  • Mitochondrial uncouplers (CCCP) or inhibitors (oligomycin) may alter SUPV3L1 distribution

  • Conditions that induce nuclear translocation (cellular stress) can be used to validate nuclear staining

  • The C-terminal region contains a potential nuclear localization signal (RRKK motif) that may be important for nuclear-mitochondrial shuttling

What are the key considerations for studying SUPV3L1 in the context of mitochondrial stress responses?

For investigating SUPV3L1 in mitochondrial stress responses:

• Stress induction protocols:

  • Oxidative stress: H₂O₂ (100-500 μM), paraquat (10-50 μM)

  • mtDNA stress: ethidium bromide (low-dose for mtDNA depletion), doxorubicin

  • Mitochondrial unfolded protein response: CCCP (5-10 μM), gamitrinib

  • Hypoxia: culture in 1-3% O₂ or CoCl₂ treatment

• Temporal analysis:

  • Monitor SUPV3L1 localization changes at multiple timepoints after stress induction

  • Acute vs. chronic stress may show different patterns of SUPV3L1 redistribution

  • Recovery phase analysis can reveal dynamics of mitochondrial RNA processing restart

• Correlation with functional outcomes:

  • Mitochondrial membrane potential measurements (TMRM, JC-1) alongside FITC-SUPV3L1 staining

  • mtRNA processing efficiency (qRT-PCR of processing intermediates)

  • Mitochondrial translation activity (puromycin incorporation)

  • Mitochondrial dynamics (fusion/fission balance)

• Disease relevance:

  • Compare stress responses between cells with wild-type and disease-associated SUPV3L1 variants

  • Assess whether SUPV3L1 localization changes correlate with disease severity or progression

  • Investigate whether pharmacological interventions that modify mitochondrial stress responses affect SUPV3L1 function in disease models