VDAC3 Antibody, FITC conjugated

What is VDAC3 and why is it important in cellular research?

VDAC3 is a voltage-dependent anion channel belonging to the mitochondrial porin family. It traverses the outer mitochondrial membrane and conducts ATP and other small metabolites across the membrane. VDAC3 is known to bind several kinases involved in intermediary metabolism and is hypothesized to form part of the mitochondrial permeability transition pore, which mediates cytochrome c release during apoptosis . Unlike other VDAC isoforms, VDAC3 plays a more specialized role in maintaining mitochondrial membrane potential and regulating cellular ATP levels, making it a key target for research into mitochondrial function and dysfunction .

What should I consider when selecting a FITC-conjugated VDAC3 antibody?

When selecting a FITC-conjugated VDAC3 antibody, consider specificity, cross-reactivity with other VDAC isoforms, host species, and validated applications. Some antibodies raised against VDAC1 may cross-react with VDAC3 but not VDAC2 . Review the antibody's validation data including Western blot, immunofluorescence, and immunoprecipitation results. For FITC-conjugated antibodies specifically, verify that the conjugation doesn't interfere with epitope binding and that the fluorophore-to-protein ratio is optimal for detection without causing aggregation. Additionally, confirm the excitation/emission spectra are compatible with your imaging system and won't cause spectral overlap with other fluorophores in your experiment .

How do I validate a VDAC3 antibody for specificity?

Validation should include multiple approaches:

• Western blot analysis comparing samples with known VDAC3 expression levels

• Testing in VDAC3 knockdown/knockout models to confirm reduction/absence of signal

• Comparative analysis with other VDAC isoform antibodies to confirm specificity

• Immunofluorescence co-localization with mitochondrial markers like MitoTracker

• Peptide competition assays to confirm epitope specificity

If working with tagged VDAC3 (e.g., FLAG-tagged), validate antibody specificity by comparing immunoprecipitation results using both the VDAC3 antibody and the epitope tag antibody . VDAC3 antibodies should show consistent localization to the mitochondrial outer membrane when examined by immunofluorescence microscopy .

What are the optimal fixation and permeabilization conditions for FITC-conjugated VDAC3 antibody in immunofluorescence?

For optimal mitochondrial VDAC3 detection with FITC-conjugated antibodies, fix cells with 2% paraformaldehyde for 15 minutes at room temperature. Permeabilize with PBS containing 0.1% Triton X-100 (PBST), followed by blocking with PBST containing 2% bovine serum albumin (BSA). FITC-conjugated antibodies typically perform best at dilutions between 1:50 and 1:200 for immunofluorescence applications . For mitochondrial co-localization studies, treat living cells with MitoTracker dye prior to fixation. This protocol preserves mitochondrial morphology while allowing sufficient antibody access to the outer mitochondrial membrane where VDAC3 resides .

How can I optimize co-immunoprecipitation protocols to study VDAC3 protein interactions?

For successful co-immunoprecipitation of VDAC3 and its interacting partners:

• Use mild lysis conditions (e.g., 1% Triton X-100 or digitonin) to preserve protein-protein interactions

• Include protease and phosphatase inhibitors in all buffers

• Pre-clear lysates with control IgG and protein G beads to reduce non-specific binding

• Use Dynabead Protein G magnetic beads pre-coated with specific antibodies against VDAC3 or its potential binding partners

• Perform reciprocal immunoprecipitations (IP with VDAC3 antibody followed by Western blotting for interacting proteins, and vice versa)

When studying specific interactions like the VDAC3-2B complex, co-transfect cells with tagged versions of both proteins (e.g., GFP-2B-Myc and N-FLAG-VDAC3) to facilitate detection and co-IP experiments . Approximately 5% of total VDACs co-immunoprecipitate under typical experimental conditions .

What controls are essential when using FITC-conjugated VDAC3 antibodies for confocal microscopy?

Essential controls include:

• Antibody specificity control: VDAC3 knockdown/knockout cells to verify signal specificity

• Secondary antibody control: Samples treated with secondary antibody only to assess non-specific binding

• Isotype control: Matched isotype antibody (rabbit IgG for rabbit-derived antibodies) to identify potential Fc receptor binding

• Autofluorescence control: Untreated fixed cells to determine background fluorescence

• Co-localization control: Mitochondrial markers (e.g., MitoTracker) to confirm proper subcellular localization

• Cross-channel bleed-through control: Single-fluorophore samples to assess spectral overlap

When examining co-localization of VDAC3 with other proteins such as viral 2B protein, include controls for each protein individually to establish baseline localization patterns .

How can FITC-conjugated VDAC3 antibodies help investigate mitochondrial ROS production?

FITC-conjugated VDAC3 antibodies can be used in conjunction with ROS-sensitive dyes to study the relationship between VDAC3 localization/abundance and ROS production. VDAC3 has been implicated in regulating reactive oxygen species (ROS) generation, particularly in viral infection contexts. Research has shown that enteroviral protein 2B interacts with VDAC3 to enhance mitochondrial ROS generation, which promotes viral replication .

To investigate this relationship:

• Use FITC-conjugated VDAC3 antibodies to monitor VDAC3 distribution and expression levels

• Combine with ROS-sensitive probes such as MitoSOX Red for superoxide detection

• Apply confocal microscopy with time-lapse imaging to track dynamic changes in VDAC3 distribution and corresponding ROS production

• Implement VDAC3 knockdown/overexpression experiments with concurrent ROS measurements to establish causality

This approach allows for precise spatiotemporal analysis of how VDAC3 distribution correlates with mitochondrial ROS production under various experimental conditions .

What is the role of VDAC3 in Parkin-mediated mitophagy and how can antibodies help elucidate this pathway?

VDAC3 has been identified as a protein that specifically interacts with Parkin on defective mitochondria and is required for efficient targeting of Parkin to mitochondria and subsequent mitophagy . To investigate this pathway:

• Use FITC-conjugated VDAC3 antibodies alongside tagged Parkin to visualize recruitment kinetics following mitochondrial damage

• Employ co-immunoprecipitation with VDAC3 antibodies to identify Parkin binding under various conditions (e.g., CCCP treatment to induce mitochondrial depolarization)

• Assess VDAC3 ubiquitination status by immunoprecipitation with anti-VDAC3 followed by immunoblotting with anti-ubiquitin antibodies

• Perform live-cell imaging to track the temporal relationship between VDAC3-Parkin interaction and mitophagy events

Experimental data suggest that approximately 5% of total VDACs co-immunoprecipitate with FLAG-Parkin under mitochondrial stress conditions, indicating this is a selective and regulated process .

How can VDAC3 antibodies be used to study its unique role in ATP/ADP regulation compared to other VDAC isoforms?

VDAC3 plays a distinct role in ATP/ADP regulation compared to VDAC1 and VDAC2. Research has shown that VDAC3 knockdown specifically decreased cellular ATP by 48% and ADP by 27%, while knockdowns of VDAC1 and VDAC2 did not significantly affect these parameters . To investigate VDAC3's unique functions:

• Use isoform-specific antibodies including FITC-conjugated VDAC3 antibodies to quantify relative expression levels of different VDAC isoforms in various cell types and conditions

• Perform selective knockdown of each VDAC isoform followed by ATP/ADP measurements

• Combine immunofluorescence with metabolic imaging techniques (e.g., FRET-based ATP sensors) to correlate VDAC3 localization with local ATP concentrations

• Investigate the distribution and phosphorylation status of VDAC3 in relation to metabolic states using phospho-specific antibodies alongside FITC-conjugated total VDAC3 antibodies

This multi-parameter approach can help distinguish the specific contributions of VDAC3 to cellular energy homeostasis that differ from other VDAC isoforms .

Why might I observe inconsistent staining patterns with FITC-conjugated VDAC3 antibodies?

Inconsistent staining patterns may result from several factors:

• Fixation artifacts: Overfixation can mask epitopes while underfixation may not preserve mitochondrial structure

• Antibody concentration: FITC-conjugated antibodies typically work best at dilutions between 1:50-1:200; concentrations outside this range may cause background or insufficient signal

• Mitochondrial dynamics: VDAC3 distribution changes with mitochondrial morphology, which is affected by cellular metabolic state

• Photobleaching: FITC is susceptible to photobleaching; use anti-fade reagents and minimize exposure

• pH sensitivity: FITC fluorescence is pH-dependent; ensure consistent buffer pH

For reliable results, optimize fixation conditions (2% paraformaldehyde for 15 minutes is often optimal), use consistent permeabilization (0.1% Triton X-100), and include MitoTracker staining as a reference for mitochondrial localization .

How can I differentiate between VDAC3 and other VDAC isoforms in my experiments?

Differentiating VDAC isoforms requires careful antibody selection and experimental design:

• Isoform-specific antibodies: Verify that your VDAC3 antibody doesn't cross-react with VDAC1 or VDAC2 through Western blotting against recombinant proteins or in knockout models

• Control samples: Include VDAC3 knockdown/knockout samples as negative controls

• Comparative analysis: Some antibodies recognize VDAC1 and VDAC3 but not VDAC2; use this selectivity pattern to distinguish isoforms

• RT-qPCR validation: Complement protein detection with isoform-specific mRNA quantification

• Mass spectrometry: For definitive identification, use immunoprecipitation followed by mass spectrometry

Be aware that commercially available anti-VDAC antibodies may have different specificity profiles. For example, the mouse monoclonal antibody 20B12AF2 recognizes VDAC1 and VDAC3 but not VDAC2 . Always verify antibody specificity in your experimental system .

How should I quantify VDAC3 colocalization with mitochondrial markers or interaction partners?

For rigorous colocalization analysis:

• Pearson's correlation coefficient: Measures the linear correlation between VDAC3 and mitochondrial marker signals (values from -1 to +1)

• Manders' overlap coefficient: Quantifies the fractional overlap between signals (values from 0 to 1)

• Intensity correlation analysis: Determines whether the intensities of two signals vary together

• Object-based colocalization: Identifies individual structures and measures their spatial relationships

When analyzing VDAC3 interactions with partners like viral protein 2B, focus on specific subcellular regions where colocalization occurs (e.g., the periphery of mitochondria) rather than whole-cell measurements . For accurate quantification, collect z-stack images with appropriate spacing (0.2-0.5 μm) and perform deconvolution before analysis to reduce out-of-focus signal .

What advanced imaging techniques can enhance VDAC3 interaction studies beyond standard confocal microscopy?

Beyond standard confocal microscopy, consider:

• Super-resolution microscopy (STED, STORM, PALM): Achieves resolution below the diffraction limit (20-50 nm), allowing visualization of VDAC3 distribution within mitochondrial subcompartments

• FRET analysis: Detects direct protein-protein interactions between VDAC3 and binding partners within 10 nm distance

• Fluorescence lifetime imaging (FLIM): Measures changes in fluorophore lifetime that occur upon protein interaction, independent of concentration

• Live-cell imaging: Tracks dynamic VDAC3 interactions in real-time using fluorescently tagged constructs

• Correlative light-electron microscopy (CLEM): Combines fluorescence localization with ultrastructural context

These techniques can reveal detailed information about VDAC3 distribution patterns and protein-protein interactions at the mitochondrial outer membrane that are not discernible with conventional microscopy .

How can I interpret changes in VDAC3 expression or localization in the context of mitochondrial function?

Interpreting VDAC3 changes requires integration with functional mitochondrial parameters:

Research has shown that VDAC3 knockdown specifically decreases cellular ATP by 48% and ADP by 27%, reducing energy charge from 0.93 to 0.89. These changes suggest that altered VDAC3 status significantly impacts mitochondrial bioenergetics . When examining VDAC3 in disease contexts or stress conditions, consider these functional correlations to properly interpret your observations.