VDAC1 Antibody, FITC conjugated

What is VDAC1 and why is it significant in research?

VDAC1 (Voltage-dependent anion-selective channel protein 1) is a mitochondrial porin that mediates the flux of metabolites and ions, thereby integrating both cell survival and death signals. It was first characterized as an outer mitochondrial membrane protein (also known as porin 31HL or porin 31HM) . VDAC1's significance stems from its dual role in cellular metabolism and mitochondria-mediated apoptosis, making it a potential therapeutic target in various neuronal disorders and cancer research . In the nervous system specifically, VDAC1 regulation has been shown to influence neuronal survival, with even subtle changes in VDAC1 levels affecting neuronal viability and causing severe alterations in retinal morphology .

What applications are VDAC1 antibodies typically used for?

VDAC1 antibodies, including FITC-conjugated versions, are primarily used in the following research applications:

• ELISA (Enzyme-Linked Immunosorbent Assay) for quantitative detection

• Immunohistochemistry (IHC) for tissue localization studies

• Western Blotting (WB) for protein expression analysis

• Tracking VDAC1 translocation between cellular compartments

• Monitoring VDAC1 oligomerization during apoptotic processes

• Investigating mitochondrial function in neuronal cells

The specific FITC-conjugation enables fluorescence detection, making these antibodies particularly useful for microscopy and flow cytometry applications where visualization of VDAC1 localization is required.

How should VDAC1 antibodies be stored for optimal performance?

For maximum stability and performance, VDAC1 antibodies require careful storage conditions:

• Store lyophilized VDAC1 antibodies at -20°C upon receipt

• For FITC-conjugated VDAC1 antibodies specifically, store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles as this significantly reduces antibody performance

• After reconstitution, VDAC1 antibodies may be stored at 4°C for one month

• For longer storage after reconstitution, aliquot and store frozen at -20°C for up to six months

• The FITC-conjugated VDAC1 antibody is typically prepared in a buffer containing 50% Glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative

These storage protocols help maintain antibody specificity and fluorescence intensity for FITC-conjugated antibodies, which is critical for reproducible experimental results.

What is the recommended antibody dilution for different experimental techniques?

Appropriate dilution factors for VDAC1 antibodies vary by application:

For VDAC1 detection in paraffin-embedded tissue sections, blocking with 10% goat serum followed by incubation with 1 μg/ml antibody has been demonstrated to be effective in both rat cardiac muscle and human cancer tissue samples .

How can VDAC1 antibodies be used to study apoptotic mechanisms?

VDAC1 antibodies provide powerful tools for investigating apoptotic mechanisms, particularly through analyzing VDAC1 oligomerization. Research has demonstrated that VDAC1 oligomerization is a critical step in mitochondria-mediated apoptosis . Methodological approaches include:

• Monitoring VDAC1 oligomerization: FITC-conjugated VDAC1 antibodies can be used in conjunction with protein cross-linking techniques to detect the formation of VDAC1 oligomers during apoptosis. Studies have shown that VDAC1 forms higher-order oligomers that facilitate cytochrome c release, a key step in the apoptotic cascade .

• VDAC1-mediated apoptosis in Bax/Bak-deficient systems: Recent research has revealed a novel mechanism of apoptosis involving VDAC1 oligomerization that functions independently of Bax and Bak, traditional mediators of mitochondrial outer membrane permeabilization. This presents a potential therapeutic avenue for cancers with downregulated Bax/Bak expression .

• VDAC1 translocation studies: Following apoptotic stimuli, VDAC1 has been observed to translocate from mitochondria to other cellular compartments including the plasma membrane and endoplasmic reticulum. FITC-conjugated antibodies allow for visualization of this translocation process through confocal microscopy .

What are the challenges in distinguishing between mitochondrial and non-mitochondrial VDAC1 pools?

VDAC1 was traditionally considered exclusively mitochondrial, but research has revealed significant non-mitochondrial pools of VDAC1, creating technical challenges for researchers:

• Subcellular fractionation approach: To accurately distinguish between mitochondrial and non-mitochondrial VDAC1, researchers should employ careful subcellular fractionation techniques followed by immunoblotting with VDAC1 antibodies. Research has demonstrated that after retinal mechanical injury, VDAC1 is upregulated specifically in the plasma membrane and endoplasmic reticulum rather than in mitochondria .

• Co-localization studies: When using FITC-conjugated VDAC1 antibodies for microscopy, co-staining with specific organelle markers (e.g., MitoTracker for mitochondria, calnexin for ER, Na+/K+-ATPase for plasma membrane) is essential to accurately determine VDAC1 localization.

• Temporal considerations: The distribution of VDAC1 can change rapidly following cellular stress. Studies have shown upregulation of non-mitochondrial VDAC1 as quickly as 2 hours after mechanical trauma , emphasizing the importance of time-course experiments.

How can researchers investigate VDAC1's role in neuronal injury and neuroprotection?

VDAC1 has emerged as a potential therapeutic target in neuronal disorders. To investigate its role in neuronal injury and potential neuroprotection:

• In vitro models of oxidative stress and mechanical injury:

  • Primary retinal cell cultures exposed to H₂O₂ (50-100 μM) can model oxidative stress

  • Treatment with VDAC1 inhibitors like DIDS (4′-diisothiocyano-2,2′-disulfonic acid stilbene) at 25 μM concentration has been shown to rescue cell viability (83.91 ± 6.81% compared to 63.66 ± 6.74% for H₂O₂ treatment alone)

  • In mechanical scratch models, DIDS treatment alters microglial morphology and astrogliosis response, demonstrating VDAC1's impact on glial cell recruitment and activation

• In vivo models using retinal trauma:

  • Mechanical trauma can be induced in rat retina using a thin needle (28-gauge)

  • VDAC1 inhibition through intraocular injection of DIDS (1 mM, 7 μL) decreases apoptosis and prevents microglial polarization

  • For VDAC1 knockdown studies, morpholino antisense oligonucleotides can be used (e.g., 5′-ATATGTGGGAGGCACAGCCATGTTC-3′) via subretinal space injection (5 μL of 0.5 mM solution)

• Analysis of inflammatory response:

  • Multiplex evaluation of cytokines has shown that VDAC1 inhibition disorganizes the inflammatory response shortly after lesion (2 hours), corresponding with the rapid regulation of VDAC1

What experimental controls are critical when using VDAC1 antibodies in apoptosis research?

When studying VDAC1's role in apoptosis, several critical controls must be incorporated:

• Specificity controls:

  • Include VDAC1 knockdown samples (using siRNA or morpholino oligonucleotides) to validate antibody specificity

  • When possible, use multiple VDAC1 antibodies targeting different epitopes to confirm findings

• Positive controls for apoptosis induction:

  • Include established apoptosis inducers (e.g., cisplatin, TRAIL, endostatin) that have been demonstrated to work through VDAC1-dependent mechanisms

  • Studies have shown that siRNA-mediated down-expression of VDAC1 prevents cisplatin-induced release of cytochrome c and apoptosis-inducing factor

• Mechanistic controls:

  • Include control experiments with anti-VDAC1 antibodies, which have been shown to prevent As₂O₃-induced cytochrome c release from isolated mitochondria

  • When studying VDAC1 oligomerization, protein cross-linking controls are essential to distinguish between experimental artifacts and genuine oligomeric forms

How should experiments be designed to study VDAC1 oligomerization?

VDAC1 oligomerization is a key event in apoptosis pathways. To effectively study this process:

• Chemical cross-linking approach:

  • Treat cells or isolated mitochondria with cell-permeable cross-linkers

  • Analyze VDAC1 oligomeric states by SDS-PAGE followed by western blotting with VDAC1 antibodies

  • Include both apoptotic stimuli and VDAC1 inhibitors like DIDS to demonstrate specificity

• FRET (Förster Resonance Energy Transfer) analysis:

  • Use FITC-conjugated VDAC1 antibodies alongside other fluorophore-conjugated VDAC1 antibodies to detect oligomerization through FRET

  • This approach allows real-time monitoring of oligomerization in living cells

• Size exclusion chromatography:

  • Analyze detergent-solubilized mitochondrial extracts to separate VDAC1 monomers from oligomers

  • Confirm oligomeric state with western blotting using VDAC1 antibodies

• Temporal considerations:

  • Design time-course experiments to capture the dynamics of VDAC1 oligomerization

  • Research has shown that VDAC1 multimers assemble rapidly after cellular injury

What methodological approaches allow discrimination between VDAC1 isoforms?

VDAC has three known isoforms (VDAC1, VDAC2, and VDAC3) with distinct functions. To specifically study VDAC1:

• Antibody selection criteria:

  • Select antibodies raised against unique epitopes of VDAC1 not present in VDAC2 or VDAC3

  • The FITC-conjugated polyclonal antibody against recombinant human VDAC1 protein (32-59AA) offers specificity for VDAC1

• Validation through knockout/knockdown models:

  • Confirm antibody specificity using VDAC1-specific knockdown (siRNA or morpholino antisense)

  • Include controls with VDAC2 and VDAC3 knockdowns to verify lack of cross-reactivity

• Mass spectrometry confirmation:

  • For critical experiments, consider validating VDAC1 identity through immunoprecipitation followed by mass spectrometry

  • This provides unambiguous identification of the specific VDAC isoform

How can researchers troubleshoot non-specific binding when using VDAC1 antibodies?

Non-specific binding is a common challenge with antibodies. For VDAC1 antibodies:

• Optimizing blocking conditions:

  • For immunohistochemistry, 10% goat serum has been demonstrated effective for blocking prior to antibody incubation

  • For western blotting, 5% BSA in TBST is recommended

• Titration experiments:

  • Perform antibody dilution series to identify optimal concentration

  • Over-concentration of antibodies is a common cause of non-specific binding

• Cross-adsorption:

  • If cross-reactivity with other VDAC isoforms is suspected, consider pre-adsorbing the antibody with recombinant VDAC2 and VDAC3 proteins

• Epitope retrieval optimization:

  • For tissue sections, heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has been shown to improve specificity

  • Test multiple retrieval buffers and conditions to optimize signal-to-noise ratio

How should researchers interpret conflicting data on VDAC1 localization?

VDAC1 has been reported in multiple cellular compartments, leading to potentially contradictory findings:

• Compartment-specific functions:

  • VDAC1 in mitochondria primarily regulates metabolite flux and initiates apoptosis

  • Plasma membrane VDAC1 may have distinct functions related to cellular signaling

  • Endoplasmic reticulum VDAC1 has been implicated in calcium homeostasis

• Reconciling contradictory data:

  • Consider cell type-specific differences in VDAC1 distribution

  • Evaluate experimental conditions, as VDAC1 localization can change rapidly following stress

  • In retinal trauma models, VDAC1 was upregulated in the plasma membrane and endoplasmic reticulum rather than mitochondria within 2 hours of injury

• Technical considerations:

  • Different fixation methods can affect antibody accessibility to various subcellular compartments

  • Confocal microscopy with z-stack analysis provides more reliable localization data than conventional fluorescence microscopy

What are the current limitations in understanding VDAC1's role in neurodegeneration?

Despite progress, several knowledge gaps remain in VDAC1 neurodegeneration research:

• Causality versus correlation:

  • While VDAC1 upregulation correlates with neurodegeneration, establishing direct causality remains challenging

  • Knockdown studies have shown that even subtle changes in VDAC1 levels affect neuronal survival and cause severe alterations in retinal morphology

• Isoform-specific functions:

  • The relative contributions of VDAC1 versus VDAC2 and VDAC3 in neurodegeneration are not fully elucidated

  • Different isoforms may have compensatory or antagonistic functions

• Interaction with disease-specific proteins:

  • VDAC1's interaction with proteins implicated in specific neurodegenerative diseases (e.g., α-synuclein, tau, amyloid-β) requires further investigation

  • These interactions may represent therapeutic targets

• Translational challenges:

  • While VDAC1 inhibitors like DIDS show neuroprotective effects in experimental models , translating these findings to clinically viable therapies faces significant hurdles

  • Selective inhibition of VDAC1 in specific cellular compartments represents a particular challenge

How does VDAC1's role in apoptosis relate to its potential as a therapeutic target?

VDAC1's dual role in metabolism and apoptosis creates both opportunities and challenges for therapeutic targeting:

• Targeting strategies:

  • Inhibition of VDAC1 oligomerization rather than general VDAC1 inhibition may provide selective anti-apoptotic effects without disrupting metabolic functions

  • DIDS has been shown to rescue cell viability in oxidative stress models by inhibiting VDAC1 oligomerization

• Context-dependent interventions:

  • In neuronal injury, VDAC1 inhibition appears neuroprotective by preventing excessive apoptosis

  • In cancer, promoting VDAC1-mediated apoptosis may be beneficial, particularly in tumors with Bax/Bak downregulation

• Therapeutic window considerations:

  • Complete VDAC1 knockdown causes severe cellular dysfunction, suggesting a narrow therapeutic window

  • Experimental models show that fine regulation of VDAC1 is critical, as even subtle changes in VDAC1 levels affect cellular survival

What emerging techniques might enhance VDAC1 research?

Several cutting-edge methodologies show promise for advancing VDAC1 research:

• Super-resolution microscopy:

  • Techniques like STORM or PALM can provide nanoscale resolution of VDAC1 distribution and oligomerization

  • When combined with FITC-conjugated antibodies, these approaches could reveal previously undetectable structural details

• In situ proximity ligation assays:

  • These techniques can reveal VDAC1 interactions with other proteins in fixed cells or tissues

  • Particularly valuable for studying VDAC1's associations with Bcl-2 family proteins and other apoptosis regulators

• CRISPR-Cas9 genome editing:

  • Generation of endogenously tagged VDAC1 can overcome limitations of antibody-based detection

  • Creation of domain-specific mutants can help dissect VDAC1's multifunctional nature

• Single-cell transcriptomics and proteomics:

  • These approaches can reveal cell type-specific variations in VDAC1 expression and regulation

  • Particularly relevant for heterogeneous tissues like retina and brain where cellular responses to injury vary significantly

How might VDAC1 research contribute to personalized medicine approaches?

VDAC1 research has several potential applications in personalized medicine:

• Biomarker development:

  • VDAC1 expression levels and oligomerization state could serve as biomarkers for disease progression in neurodegeneration

  • Variations in VDAC1 response to inhibitors might predict treatment efficacy

• Targeted therapies:

  • Patients with specific VDAC1 polymorphisms might respond differently to VDAC1-targeting compounds

  • In cancers with Bax/Bak downregulation, VDAC1-induced apoptosis offers a novel approach for targeted tumor therapies

• Combination treatment strategies:

  • VDAC1 inhibitors like DIDS could potentially be combined with existing neuroprotective agents

  • VDAC1-targeted therapies might sensitize resistant tumors to conventional chemotherapeutics