VDAC3 Antibody, Biotin conjugated

What is VDAC3 and why are biotin-conjugated antibodies useful for its detection?

VDAC3 belongs to the eukaryotic mitochondrial porin family. It forms a channel through the mitochondrial outer membrane that allows diffusion of small hydrophilic molecules . Biotin-conjugated VDAC3 antibodies offer significant advantages for detection protocols due to the strong and specific interaction between biotin and streptavidin, enabling enhanced signal amplification in detection systems. These antibodies are particularly valuable for pull-down assays, immunoprecipitation experiments, and detection protocols requiring high sensitivity or multiple layering techniques .

What applications are most suitable for biotin-conjugated VDAC3 antibodies?

Biotin-conjugated VDAC3 antibodies are highly suitable for:

The versatility of these antibodies stems from the tight biotin-streptavidin binding and the ability to couple with various detection systems including fluorescent, enzymatic, and gold-based methods .

What dilution ranges are recommended for different applications?

Based on validated protocols across multiple research publications, the following dilution ranges are recommended:

It is strongly recommended that researchers titrate the antibody in each testing system to obtain optimal results, as sensitivity can vary between detection methods and sample types .

How do storage conditions affect biotin-conjugated VDAC3 antibody performance?

Proper storage is critical for maintaining antibody functionality. Biotin-conjugated VDAC3 antibodies should be stored at -20°C, where they remain stable for approximately one year. After reconstitution, antibodies can be stored at 4°C for about one month, but for longer periods, aliquoting and freezing at -20°C is recommended .

Key considerations include:

• Avoid repeated freeze-thaw cycles which can cause protein denaturation and biotin degradation

• For most commercial preparations, aliquoting is unnecessary for -20°C storage

• Some preparations (typically 20μl sizes) contain 0.1% BSA as a stabilizer

• Storage buffers generally contain PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

What are the major cross-reactivity considerations when using VDAC3 antibodies?

When selecting a VDAC3 antibody for research, cross-reactivity with other VDAC isoforms (VDAC1 and VDAC2) is a critical consideration. Human VDAC isoforms share significant sequence homology but differ notably in their cysteine content: VDAC1 has two cysteines, VDAC2 has nine, and VDAC3 has six .

Researchers should:

• Verify isoform specificity through validation data

• Consider testing antibodies against knockout/knockdown controls

• Be aware that some antibodies deliberately target multiple VDAC isoforms (like anti-VDAC1/Porin + VDAC3 antibody [20B12AF2])

• Check the immunogen sequence to identify potential cross-reactivity with other mitochondrial proteins

What optimization steps are needed for using biotin-conjugated VDAC3 antibodies in immunohistochemistry?

Optimization for IHC requires attention to several parameters:

• Antigen retrieval: For VDAC3 detection in tissues, TE buffer pH 9.0 is suggested as the primary method, with citrate buffer pH 6.0 as an alternative

• Blocking optimization: Due to endogenous biotin in tissues (particularly liver and kidney), use biotin/avidin blocking kits to reduce background

• Dilution optimization: Begin with 1:50-1:500 range and adjust based on signal-to-noise ratio

• Positive controls: Use tissues with confirmed VDAC3 expression (human liver cancer tissue, human osteosarcoma tissue)

• Counterstaining: Adjust timing to ensure nuclear details are visible without obscuring VDAC3 staining in mitochondria

How can biotin-conjugated VDAC3 antibodies be used in studying protein-protein interactions in mitochondrial research?

Biotin-conjugated VDAC3 antibodies offer powerful tools for investigating protein-protein interactions through various methodologies:

• Co-immunoprecipitation (Co-IP): Using streptavidin-coated beads to pull down biotin-tagged VDAC3 complexes, researchers can identify interaction partners. This approach has revealed associations between VDAC3 and proteins involved in oxidative stress response (GSTO-1, PRDX, GSTK-1) and protein quality control pathways .

• Proximity-based labeling: Combining biotin-conjugated antibodies with crosslinking agents allows identification of transient or weak interactions in the mitochondrial membrane environment.

• Pull-down assays with site-directed mutations: This approach has been used to map specific interaction domains, revealing, for example, how dankastatin B interacts with cysteine C65 of VDAC3 .

• Chemoproteomics: As demonstrated in studies with dankastatin B, biotin-conjugated probes can be used to identify covalent modifications of VDAC3 in living cells .

What considerations are important when using VDAC3 antibodies to study oxidative modifications of the protein?

VDAC3 contains six cysteine residues that undergo various oxidative modifications, making it a potential sensor of mitochondrial redox status. When studying these modifications:

• Sample preparation: Avoid introducing artificial oxidation during sample preparation by including reducing agents and alkylating reagents appropriately

• Detection specificity: Consider using antibodies that specifically recognize different oxidation states (e.g., -SH, -SOH, -SO₂H, -SO₃H) of cysteine residues

• Evolutionary conservation: Note that cysteine residues in VDAC3 follow an evolutionarily conserved redox modification pattern that may reflect their position relative to the cytosol or intermembrane space

• Physiological relevance: VDAC3's cysteines are never detected as totally oxidized, suggesting they undergo continuous oxidation-reduction cycles that may regulate intracellular ROS levels

• Combined approaches: Use mass spectrometry alongside immunological detection to precisely characterize the type and location of oxidative modifications

What protocols exist for using biotin-conjugated VDAC3 antibodies in studying mitochondrial dysfunction in disease models?

Several validated protocols exist for studying mitochondrial dysfunction using biotin-conjugated VDAC3 antibodies:

• Mitochondrial quality control assessment: VDAC3 has been implicated in mitochondrial quality control mechanisms, including mitophagy. Biotin-conjugated antibodies can be used to track changes in VDAC3 ubiquitination status, which precedes mitophagy .

• Cancer research applications: In studies of anti-cancer compounds like dankastatin B, biotin-conjugated VDAC3 antibodies help identify VDAC3 as a potential therapeutic target. The protocol typically involves:

  • Treating cells with the compound of interest

  • Using activity-based protein profiling with biotin-conjugated probes

  • Analyzing binding through gel-based detection or mass spectrometry

• Redox signaling investigation: For studying VDAC3's role as a redox sensor, protocols typically include:

  • Oxidative challenge of cells (e.g., with H₂O₂ or specific inhibitors)

  • Isolation of mitochondria under non-reducing conditions

  • Detection of VDAC3 oxidation state using biotin-switch techniques combined with VDAC3-specific antibodies

How can researchers differentiate between specific and non-specific binding when using biotin-conjugated VDAC3 antibodies?

To distinguish between specific and non-specific binding:

• Include appropriate controls:

  • Isotype controls to assess background binding of antibody constant regions

  • Blocking peptide controls using the immunogenic peptide

  • Knockdown/knockout samples where VDAC3 expression is reduced or eliminated

• Address biotin-specific issues:

  • Use avidin/biotin blocking kits to reduce endogenous biotin interference

  • Include streptavidin-only controls to assess non-specific binding of the detection system

  • Consider tissue-specific endogenous biotin levels (higher in liver, kidney, brain)

• Cross-reactivity assessment:

  • Verify antibody specificity against all three VDAC isoforms

  • Use recombinant VDAC proteins to confirm specificity

  • Check for cross-reactivity with other mitochondrial outer membrane proteins

What are the common pitfalls in interpreting data from experiments using biotin-conjugated VDAC3 antibodies?

Common interpretation pitfalls include:

• Misattribution of cellular localization: VDAC3 is primarily a mitochondrial outer membrane protein, but has been detected in other locations like the plasma membrane. Confirm localization with mitochondrial markers.

• Overlooking redox state influence: VDAC3's multiple cysteine residues undergo various oxidative modifications that can affect antibody binding. Consider whether experimental conditions alter the redox environment.

• Isoform confusion: The three VDAC isoforms have different expression patterns across tissues. Validate that observed signals correspond to VDAC3 rather than other isoforms.

• Biotin interference: Endogenous biotin can create false positives, particularly in biotin-rich tissues or when cells are cultured with biotin-containing media.

• Molecular weight misinterpretation: VDAC3's calculated molecular weight is 31 kDa, but it may migrate differently (often observed at 30 kDa) depending on gel conditions and post-translational modifications .

How can conflicting results between different detection methods using VDAC3 antibodies be reconciled?

When faced with conflicting results across different detection methods:

• Consider epitope accessibility: The three-dimensional structure of VDAC3 may expose different epitopes depending on the technique. Biotin conjugation can also affect antibody binding characteristics.

• Evaluate sample preparation effects: Different preparation methods can alter protein conformation or expose different epitopes:

  • Denaturing conditions (like SDS-PAGE) versus native conditions

  • Fixation effects in IHC and IF applications

  • Membrane solubilization methods for co-IP experiments

• Assess sensitivity thresholds: Methods vary in detection sensitivity:

  • Western blot can detect lower protein abundance than IHC

  • ELISA typically offers quantitative data over a wider dynamic range

  • Flow cytometry provides single-cell resolution but may have higher threshold requirements

• Verify with orthogonal approaches: Combine antibody-based detection with:

  • Mass spectrometry for protein identification

  • RT-PCR for transcript-level validation

  • CRISPR/Cas9 gene editing for specificity control

How do the oxidation states of VDAC3 cysteines affect antibody binding and experimental outcomes?

VDAC3 contains six cysteine residues that can exist in various oxidation states, potentially affecting antibody recognition:

• The oxidation state of these cysteines is physiologically regulated and can be altered by cellular redox conditions, affecting protein conformation and epitope accessibility.

• Research has shown that VDAC3's cysteines follow an evolutionarily conserved pattern of oxidation, which may reflect their functional importance in redox sensing.

• Experimental considerations should include:

  • Using reducing or non-reducing conditions during sample preparation depending on the research question

  • Being aware that fixation methods can alter the redox state of proteins

  • Considering whether the antibody's epitope includes or is conformationally affected by cysteine residues

  • Validating findings with antibodies targeting different epitopes of VDAC3

What methodological approaches can resolve contradictions in published data regarding VDAC3 function in mitochondrial quality control?

To address contradictions in the literature regarding VDAC3's role in mitochondrial quality control:

• Combined genetic and pharmacological approaches: Use both VDAC3 knockdown/knockout models and specific inhibitors to distinguish between direct effects and compensatory mechanisms.

• Tissue and cell-type specific analyses: VDAC3 function may differ between tissues and cell types; systematic comparison using the same methodologies can resolve apparent contradictions.

• Temporal dynamics investigation: Many contradictions arise from studying processes at different time points; time-course experiments with biotin-conjugated VDAC3 antibodies can reveal dynamic changes.

• Post-translational modification mapping: Comprehensive analysis of VDAC3 modifications (oxidation, phosphorylation, ubiquitination) across experimental conditions can explain functional differences.

• Interactome analysis under defined conditions: Using biotin-conjugated antibodies for pull-down assays under standardized conditions can reveal context-dependent protein interactions that explain functional variations .

How can biotin-conjugated VDAC3 antibodies be integrated with emerging technologies for studying mitochondrial membrane dynamics?

Integration of biotin-conjugated VDAC3 antibodies with emerging technologies offers new research possibilities:

• Super-resolution microscopy: Combining biotin-conjugated VDAC3 antibodies with streptavidin-conjugated quantum dots or organic fluorophores enables visualization of VDAC3 distribution and clustering at nanoscale resolution.

• Live-cell imaging: Though challenging with antibodies, cell-permeable biotin-conjugated nanobodies or mini-antibodies against VDAC3 could enable dynamic studies of mitochondrial outer membrane proteins.

• Proximity labeling techniques: Biotin-conjugated antibodies can be used with proximity labeling enzymes (BioID, APEX) to identify proteins within the VDAC3 microenvironment under different conditions.

• Cryo-electron tomography: Biotin-conjugated antibodies with gold nanoparticle-labeled streptavidin can serve as fiducial markers for aligning tomograms and localizing VDAC3 in its native membrane environment.

• Single-molecule tracking: Using streptavidin-conjugated fluorophores with biotin-tagged anti-VDAC3 antibody fragments enables tracking of individual VDAC3 molecules in the mitochondrial membrane under physiological conditions .