VDAC1 Antibody, HRP conjugated

What is VDAC1 and why is it important in cell biology research?

VDAC1 (Voltage-dependent anion-selective channel protein 1) is a protein primarily located in the outer mitochondrial membrane, although it can also be found in the plasma membrane. It forms a channel through these membranes and plays critical roles in cellular metabolism and apoptosis. VDAC1 functions as the outer mitochondrial membrane receptor for hexokinase and BCL2L1, mediating the cross-talk between mitochondria and cytoplasm . It is involved in small molecule diffusion, cell volume regulation, and apoptotic pathways . VDAC1 may participate in the formation of the permeability transition pore complex (PTPC), which is responsible for the release of mitochondrial products that trigger apoptosis .

In research contexts, VDAC1 serves as a valuable target for studying mitochondrial function, cellular energy metabolism, and cell death mechanisms. The protein's central role in both normal cellular physiology and pathological conditions makes it particularly relevant for cancer research, neurodegenerative disease studies, and investigations into cellular metabolic regulation .

What are the key features of HRP-conjugated VDAC1 antibodies?

HRP (Horseradish Peroxidase)-conjugated VDAC1 antibodies offer several advantages for research applications:

Most commercially available HRP-conjugated VDAC1 antibodies are purified by Protein G or Protein A affinity chromatography and are formulated in a buffer containing PBS (pH 7.4), glycerol (50%), and sodium azide (0.1%) for optimal stability . These antibodies recognize specific epitopes within the full-length (amino acids 1-283 or 1-293) human VDAC1 protein .

How should researchers properly store and handle HRP-conjugated VDAC1 antibodies?

Proper storage and handling are essential to maintain antibody activity and specificity:

For long-term storage (>1 month):

• Store at -20°C in small aliquots to avoid repeated freeze-thaw cycles

• Centrifuge vials briefly before opening to collect material at the bottom

• Keep original vial tightly sealed when not in use

For short-term storage:

• Store at 2-8°C (refrigerated) for up to 2-4 weeks

• Avoid exposure to direct light to preserve HRP activity

Working solutions:

• Prepare dilutions immediately before use

• Discard any unused diluted antibody

• Return stock solution to appropriate storage temperature promptly

Critical handling precautions:

• Avoid repeated freeze-thaw cycles which degrade both antibody and HRP enzyme activity

• Use clean, nuclease-free pipette tips when handling the antibody

• Wear gloves to prevent contamination and exposure to sodium azide

• Allow refrigerated antibodies to equilibrate to room temperature before opening to prevent condensation

Most HRP-conjugated VDAC1 antibodies remain stable for 12-24 months when stored properly according to manufacturer specifications .

What are the recommended dilutions for using VDAC1 antibody (HRP conjugated) in different applications?

Based on validated protocols, the following dilutions are recommended for HRP-conjugated VDAC1 antibodies:

Important considerations for dilution optimization:

• Begin with recommended dilutions and adjust based on signal intensity and background

• Perform preliminary titration experiments with positive control samples

• When switching to a new lot or brand of antibody, re-optimization is advisable

• Signal development time should be adjusted based on dilution used (longer times for higher dilutions)

• Different sample types may require different optimal dilutions

The expected molecular weight for VDAC1 detection is approximately 30 kDa . Verification of specific detection at this molecular weight is critical for confirming assay validity.

How can researchers validate the specificity of VDAC1 antibodies in their experiments?

Validating antibody specificity is crucial for reliable experimental results. For VDAC1 antibodies, the following methodological approaches are recommended:

Genetic validation approaches:

• Use samples from VDAC1 knockout mice or VDAC1-knockdown cells as negative controls

• Several VDAC1 antibodies (including clone S152B-23) have been validated using knockout models to confirm they do not cross-react with VDAC2 or VDAC3

Biochemical validation:

• Western blot analysis should show a single band at approximately 30 kDa

• Perform peptide competition assays by pre-incubating antibody with immunizing peptide

• Test for cross-reactivity with recombinant VDAC2 and VDAC3 proteins

Localization validation:

• Confirm that staining pattern matches expected mitochondrial distribution

• Co-stain with established mitochondrial markers like TOM20 or COX IV

• Compare pattern with alternative VDAC1 antibodies targeting different epitopes

Expression systems validation:

• Generate VDAC1 overexpression systems as positive controls

• Use siRNA or shRNA knockdown of VDAC1 to demonstrate specificity

• Compare staining patterns with established tissue expression profiles for VDAC1

Based on search results, antibodies raised against full-length human VDAC1 (amino acids 1-283) typically show 98% sequence identity with mouse and rat VDAC1 proteins, making them suitable for cross-species applications .

How can VDAC1 antibodies be used to investigate cancer cell metabolism and apoptosis resistance?

VDAC1 plays a central role in cancer cell metabolism and apoptosis resistance, making HRP-conjugated VDAC1 antibodies valuable tools for cancer research using the following methodological approaches:

Metabolism studies:

• Use co-immunoprecipitation with VDAC1 antibodies to isolate and characterize VDAC1-hexokinase complexes

• The interaction between hexokinases and VDAC1 provides tumor cells with a metabolic advantage through the "Warburg effect" by giving hexokinases preferential access to mitochondrially-produced ATP

• High expression of both VDAC1 and hexokinases has been observed in many cancer types, allowing cancer cells to maintain elevated glycolysis rates even under aerobic conditions

Apoptosis resistance mechanisms:

• Track VDAC1 oligomerization during apoptosis using chemical cross-linking followed by Western blotting with HRP-conjugated VDAC1 antibodies

• VDAC1 oligomeric assembly has been shown to be coupled to apoptosis induction, with oligomerization increasing substantially upon apoptosis induction and inhibited by apoptosis blockers

• Under normal conditions, VDAC1 interacts with anti-apoptotic Bcl-2 family members to inhibit apoptosis, while cellular stress triggers the activation of the intrinsic apoptotic pathway

Experimental applications:

• Study how VDAC1-interacting molecules (VA molecules) affect cancer cell viability by competing with NADH for binding to VDAC1

• These VA molecules have been identified to selectively bind VDAC1 and display specificity toward cancer cells

• Recent studies demonstrated that VDAC1 antagonists can reduce cell proliferation in cancer cells by causing mitochondrial distress through displacement of NADH from VDAC1

• Examine oligomeric status of cellular VDAC1 under physiological versus apoptotic conditions using conformation-specific antibodies

This research is particularly valuable given that resistance of cancer cells to apoptosis often involves quenching the mitochondrial apoptotic pathway through over-expression of anti-apoptotic proteins that interact with VDAC1 .

What experimental approaches can distinguish between VDAC1, VDAC2, and VDAC3 when studying mitochondrial functions?

Distinguishing between VDAC isoforms is crucial for understanding their specific roles in mitochondrial functions. The following approaches enable selective analysis:

Isoform-specific antibodies:

• Use validated antibodies like clone S152B-23 that have been confirmed not to cross-react with VDAC2 or VDAC3 based on knockout validation

• The specificity is critical as VDAC1 shares >60% sequence identity with VDAC2 and VDAC3

• Western blotting should be performed to confirm isoform specificity using recombinant proteins of all three isoforms

Genetic manipulation strategies:

• Design isoform-specific siRNAs or shRNAs targeting unique regions of each VDAC isoform

• Selective silencing of endogenous hVDAC1 has been achieved using an shRNA-expressing vector targeting nucleotides 483-501 of the hVDAC1 coding sequence

• Create CRISPR/Cas9-mediated knockout cell lines for each VDAC isoform separately

Structural and functional analysis:

• Structure-based analysis reveals unique properties of VDAC1 oligomerization sites

• The predicted weakly stable β-strands (particularly β-strands 1, 2, and 19) represent interfaces between VDAC1 monomers in oligomeric structures

• Analysis of crystal packing revealed an anti-parallel dimer that further assembles into hexamers in mouse VDAC1, while NMR-based structure of recombinant human VDAC1 implied formation of a parallel dimer

Isoform-specific detection in plants:

• For plant research, antibodies recognizing all VDAC isoforms in plants are available

• Plant VDAC proteins show expected molecular weight around 29 kDa (for Arabidopsis thaliana)

• Anti-VDAC1-5 antibodies recognize conserved epitopes across multiple plant VDAC proteins with confirmed reactivity in various plant species

These approaches enable researchers to discriminate between VDAC isoforms and study their specific roles in mitochondrial function, apoptosis regulation, and energy metabolism.

How can VDAC1 antibodies be used to study VDAC1-hexokinase interactions in the context of the Warburg effect?

The VDAC1-hexokinase interaction is a key component of the Warburg effect in cancer metabolism. HRP-conjugated VDAC1 antibodies can be employed in several methodological approaches to study this interaction:

Interaction assessment methodologies:

• Co-immunoprecipitation: Use VDAC1 antibodies to pull down VDAC1 and associated hexokinase isoforms (HK-I and HK-II)

• Proximity ligation assays: Combine VDAC1 and hexokinase antibodies to visualize and quantify in situ interactions

• Competition studies: Test VDAC1-derived peptides for their ability to disrupt the VDAC1-hexokinase interaction

Functional consequences analysis:

• The interaction between hexokinases and VDAC1 has a dual effect in cancer cells:

  • It prevents VDAC1 interaction with pro-apoptotic factors

  • It provides tumor cells with metabolic advantages through preferential access to mitochondrial ATP

• VDAC1-based peptides have been shown to detach hexokinase from VDAC1, leading to decreased cellular ATP levels and triggering apoptotic cell death

Experimental design considerations:

• Manipulate VDAC1-hexokinase interactions using specific inhibitors or VDAC1-derived peptides

• Monitor metabolic consequences through measurements of glycolytic flux, mitochondrial respiration, and ATP production

• Use immunohistochemistry with HRP-conjugated VDAC1 antibodies on cancer tissue microarrays to correlate expression patterns with clinical parameters

Clinical relevance:

• VDAC1-based peptides (Antp-LP4 and N-Terminal-Antp) have been shown to selectively kill peripheral blood mononuclear cells from chronic lymphocytic leukemia patients while sparing those from healthy donors

• The cell death induction by these peptides correlates with detachment of hexokinase, release of cytochrome c, loss of mitochondrial membrane potential, and decreased cellular ATP levels

These approaches provide valuable insights into how cancer cells exploit the VDAC1-hexokinase interaction to promote survival and metabolic reprogramming, potentially leading to novel therapeutic strategies.

What methodological considerations are important for studying VDAC1 oligomerization during apoptosis?

Investigating VDAC1 oligomerization during apoptosis presents several technical challenges requiring specific methodological solutions:

Stabilization of oligomeric complexes:

• Use chemical cross-linking agents to capture transient oligomeric states before cell lysis

• Structure-based and computation-based selection of predicted VDAC1 dimerization sites, combined with site-directed mutagenesis and chemical cross-linking, have successfully identified contact sites between VDAC1 molecules in dimers and higher oligomers

• The predicted weakly stable β-strands were experimentally confirmed to represent the interfaces between VDAC1 monomers in oligomeric structures

Structural approaches:

• Analysis using symmetry operators on the NMR-based structure of recombinant human VDAC1 implied formation of a parallel dimer

• Crystal packing analysis of mouse VDAC1 revealed an anti-parallel dimer that further assembles into hexamers

• In both human and mouse VDAC1, the dimer interface is formed by β-strands 1, 2, 18, and 19 and potentially extends to include β-strands 3 and 4

Mutation-based strategies:

• Replace hydrophobic amino acids with charged residues in β-strands 1, 2, and 19 to interfere with VDAC1 oligomerization

• Introduce cysteine residues at potential contact sites to enable disulfide cross-linking

• Verify oligomerization status using HRP-conjugated VDAC1 antibodies in Western blotting

Detection approaches:

• Use non-denaturing extraction methods with mild detergents to preserve native oligomeric complexes

• Perform blue native PAGE followed by Western blotting with HRP-conjugated VDAC1 antibodies

• Apply time-course experiments to track oligomerization during apoptosis progression

Research has shown that VDAC1 oligomerization increases substantially upon apoptosis induction and is inhibited by apoptosis blockers . This oligomerization process is recognized as a critical step in mitochondria-mediated apoptosis, mediating the release of apoptotic proteins such as cytochrome c .

How are VDAC1 antibodies utilized in developing VDAC1-targeted therapies for cancer?

VDAC1 antibodies play essential roles in the development and validation of VDAC1-targeted therapies for cancer:

Target validation and expression profiling:

• Use HRP-conjugated VDAC1 antibodies for Western blotting and immunohistochemistry to quantify VDAC1 expression across cancer types

• Compare expression levels in tumor versus normal tissues to identify cancers with elevated VDAC1 expression

• Recent studies have identified small molecules (VA molecules) that selectively bind to VDAC1 and display specificity toward cancer cells

Therapeutic compound characterization:

• VDAC1-based peptides and small molecules have been designed to target VDAC1 and its interactions

• VA molecules (VDAC Antagonists) are characterized by a three-ring architecture and specifically bind VDAC1 in a defined pocket that partially overlaps with the NADH binding site

• These molecules compete with NADH in a dose-response manner, resulting in lower mitochondrial oxygen consumption, suggesting mitochondrial distress

Mechanism of action studies:

• VDAC1 antibodies help determine how compounds affect VDAC1 interactions with anti-apoptotic proteins

• VDAC1-based cell-penetrating peptides (Antp-LP4 and N-Terminal-Antp) can target anti-apoptotic proteins to prevent their pro-survival activities

• These peptides induce apoptosis by activating the mitochondria-mediated pathway, reflected in membrane blebbing, condensation of nuclei, DNA fragmentation, release of mitochondrial cytochrome c, and loss of mitochondrial membrane potential

Selectivity assessment:

• VDAC1-based peptides have demonstrated selective cytotoxicity against cancer cells

• Studies show these peptides selectively kill peripheral blood mononuclear cells obtained from chronic lymphocytic leukemia patients while sparing those from healthy donors

• Experiments on organoids derived from intrahepatic cholangiocarcinoma patients demonstrated a dose-dependent reduction in cell viability upon treatment with VA molecules, with lower impact on healthy cells than conventional treatments like gemcitabine

These approaches illustrate how VDAC1 antibodies facilitate the development of novel cancer treatment strategies by enabling precise targeting of cancer-specific metabolic vulnerabilities.

What applications are validated for HRP-conjugated VDAC1 antibodies?

HRP-conjugated VDAC1 antibodies have been validated for multiple research applications:

HRP-conjugated VDAC1 antibodies detect a specific band at approximately 30 kDa in Western blots . Several antibodies, like clone S152B-23, have been extensively validated and do not cross-react with VDAC2 or VDAC3 based on knockout validation results .

For plant research, specialized anti-VDAC1-5 antibodies recognizing multiple plant VDAC isoforms are available in both unconjugated and HRP-conjugated formats .

What controls should be included when using VDAC1 antibodies in experimental protocols?

Proper controls are essential for reliable results with VDAC1 antibodies:

Positive controls:

• Tissues/cells known to express VDAC1 (brain, liver, heart tissues)

• Recombinant VDAC1 protein as a standard for Western blots

• Mitochondrial fractions as positive controls for subcellular localization studies

Negative controls:

• VDAC1 knockout or knockdown samples (when available)

• Secondary antibody-only controls to assess background staining

• Isotype controls (matching the primary antibody's isotype) to evaluate non-specific binding

Specificity controls:

• Peptide competition assays where the antibody is pre-incubated with the immunizing peptide

• Cross-reactivity assessment with related proteins (VDAC2, VDAC3)

• Several commercially available VDAC1 antibodies specifically do not cross-react with VDAC2 or VDAC3 based on knockout validation results

Subcellular localization controls:

• Co-staining with established mitochondrial markers for colocalization studies

• Fractionation controls to verify mitochondrial enrichment

• Nuclear and cytosolic markers to exclude contamination in fractionation experiments

Experimental condition controls:

• Untreated or vehicle-treated samples when studying effects of treatments

• Time course controls when studying temporal changes

• Treatment with known VDAC1-modulating compounds (e.g., VDAC1-based peptides) as positive controls for functional studies

Implementing these controls ensures confidence in experimental findings and helps distinguish specific signals from artifacts or background.

What are the challenges and solutions for multiplex immunoassays using HRP-conjugated VDAC1 antibodies?

Multiplex immunoassays with HRP-conjugated VDAC1 antibodies present specific challenges requiring technical solutions:

Signal differentiation challenges:

• HRP produces a single signal type that cannot be spectrally distinguished from other HRP signals

• This limits direct multiplexing with other HRP-conjugated antibodies

Strategic approaches for multiplexing:

• Combine HRP-conjugated VDAC1 antibodies with differently labeled antibodies (e.g., alkaline phosphatase for other targets)

• Alternative VDAC1 antibody conjugates are available, including FITC, FL650, biotin, and APC

• Use tyramide signal amplification (TSA) with spectrally distinct fluorophores for sequential detection

Optimization strategies:

• Carefully titrate antibody dilutions to minimize background (1:1000 for WB, 1:5000 for IHC)

• Refine blocking protocols to prevent non-specific binding with multiple antibodies

• Determine optimal sequence for applying multiple antibodies (generally detect less abundant proteins first)

Signal development considerations:

• Select appropriate HRP substrates based on detection method and multiplexing needs

• Control enzymatic reaction times to prevent signal oversaturation

• Include quantification standards for accurate multi-parameter analysis

Quality control measures:

• Always run single-antibody controls alongside multiplex assays

• Verify that multiplex signal patterns match single-staining experiments

• Confirm critical findings with alternative detection methods

These technical considerations enable successful incorporation of HRP-conjugated VDAC1 antibodies into multiplex assays for comprehensive analysis of VDAC1 and its interactions in complex biological systems.

How are VDAC1 antibodies used in cancer research?

VDAC1 antibodies have multiple applications in cancer research focused on both basic mechanisms and therapeutic development:

Mechanistic investigations:

• Characterizing altered VDAC1 expression in cancer cells through Western blotting and immunohistochemistry

• High expression of VDAC1 has been observed in many cancer types, correlating with their high metabolic demands

• Studying VDAC1-hexokinase interactions, which provide cancer cells with metabolic advantages through the Warburg effect

• Examining VDAC1 oligomerization status in cancer cells versus normal cells using chemical cross-linking followed by Western blotting

Therapeutic development:

• Screening and characterizing VDAC1-targeting compounds

• VA molecules (VDAC Antagonists) that compete with NADH for binding to VDAC1 have shown cancer-selective effects

• These molecules caused mitochondrial distress and reduced cell proliferation in cancer cells compared to non-cancerous cells

• When tested on organoids derived from intrahepatic cholangiocarcinoma patients, VA molecules showed a dose-dependent reduction in cell viability with lower impact on healthy cells than conventional treatments

Apoptosis resistance mechanisms:

• VDAC1-based peptides target anti-apoptotic proteins to prevent their pro-survival activities

• These peptides selectively kill cancer cells while sparing normal cells

• The selective killing correlates with detachment of hexokinase, release of cytochrome c, loss of mitochondrial membrane potential, and decreased cellular ATP levels

Clinical correlations:

• Using HRP-conjugated VDAC1 antibodies for immunohistochemistry on cancer tissue microarrays

• Correlating VDAC1 expression patterns with clinical parameters and treatment response

• VDAC1-based peptides have shown selective cytotoxicity against chronic lymphocytic leukemia cells compared to normal cells

These applications highlight the central role of VDAC1 in cancer metabolism and survival, positioning it as both a biomarker and therapeutic target.

What role do VDAC1 antibodies play in studying neurodegenerative diseases?

VDAC1 antibodies are valuable tools for investigating neurodegeneration, particularly focusing on mitochondrial dysfunction and protein interactions:

SOD1-VDAC1 interactions in ALS:

• VDAC1 antibodies have been used to study interactions between VDAC1 and superoxide dismutase 1 (SOD1), relevant to amyotrophic lateral sclerosis (ALS)

• Recent studies identified the SOD1 sequence interacting with VDAC1, which is localized on the protein surface

• VDAC1-derived peptides rescued mutant SOD1-induced toxicity in neuronal cells

• Co-immunoprecipitation with VDAC1 antibodies helped characterize these protein interactions

Mitochondrial dysfunction assessment:

• VDAC1 antibodies enable quantification of VDAC1 expression levels in affected brain regions

• Immunohistochemistry using HRP-conjugated VDAC1 antibodies can reveal alterations in mitochondrial distribution

• Co-localization studies with other mitochondrial markers help assess mitochondrial integrity

Oligomerization and apoptosis:

• Neurodegenerative diseases often involve dysregulated apoptosis

• VDAC1 oligomerization status can be monitored using chemical cross-linking followed by Western blotting with VDAC1 antibodies

• This approach helps understand the role of mitochondria-mediated apoptosis in neurodegeneration

Therapeutic targeting:

• VDAC1 antibodies facilitate screening of compounds that modulate VDAC1 function or interactions

• VDAC1-derived peptides have shown neuroprotective effects by interfering with pathological protein interactions

• The N-terminal peptide with the last six C-terminal residues removed (Δ21-26) lost cell death activity, suggesting potential therapeutic applications

These applications highlight VDAC1's importance in neuronal health and disease processes, positioning it as a potential therapeutic target for neurodegenerative disorders.

What are the emerging research directions for VDAC1 antibody applications?

Several exciting research directions are emerging for VDAC1 antibody applications:

Advanced therapeutic development:

• Design and validation of next-generation VDAC1-targeting compounds

• Over 27 versions of cell-penetrating VDAC1-based peptides have been designed and screened for optimal therapeutic properties

• Development of companion diagnostics using VDAC1 antibodies to identify patients likely to respond to VDAC1-targeted therapies

• Exploration of VA molecules as chemical entities representing promising candidates for further optimization and development as cancer therapy strategies

Novel technical approaches:

• Development of conformation-specific antibodies that recognize specific oligomeric states of VDAC1

• Application of super-resolution microscopy with VDAC1 antibodies to visualize mitochondrial dynamics at nanoscale resolution

• Integration of VDAC1 antibodies into high-throughput screening platforms for drug discovery

Expanded disease applications:

• Investigation of VDAC1's role in metabolic disorders and diabetes

• Exploration of VDAC1 as a biomarker for mitochondrial dysfunction in aging

• Studies of VDAC1 in immune cell function and inflammatory diseases

Structure-function relationships:

• Use of VDAC1 antibodies to validate structural models and interaction sites

• Combination of cryo-EM, X-ray crystallography, and antibody-based validation

• Structure-guided design of specific modulators of VDAC1 function

The continued refinement and diversification of VDAC1 antibodies, including various conjugation options (HRP, fluorescent dyes, nanoparticles), will further expand their utility in both basic research and translational applications, driving discoveries in mitochondrial biology, cellular metabolism, and therapeutic development.

What resources are available for researchers working with VDAC1 antibodies?

Researchers working with VDAC1 antibodies have access to a variety of resources:

Commercial antibody options:

• Multiple validated VDAC1 antibodies are available with various conjugations (HRP, FITC, FL650, biotin)

• Clone S152B-23 is a widely validated mouse monoclonal antibody that does not cross-react with VDAC2 or VDAC3

• Plant-specific VDAC antibodies recognizing all VDAC isoforms in plants are available for plant research

Structural resources:

• NMR-based structure of recombinant human VDAC1 and crystal structure of mouse VDAC1 provide insights into dimerization interfaces

• Structural data identifies β-strands 1, 2, 18, and 19 as critical for VDAC1 oligomerization

• Molecular models of VDAC1 interactions with hexokinase, Bcl-2 family proteins, and small molecules

Experimental tools:

• VDAC1 knockout and knockdown systems for antibody validation

• Recombinant VDAC1 protein standards for quantitative analyses

• VDAC1-derived peptides as research tools for disrupting specific protein interactions

Published protocols:

• Validated protocols for Western blotting, immunohistochemistry, and immunofluorescence using HRP-conjugated VDAC1 antibodies

• Methods for studying VDAC1 oligomerization using chemical cross-linking

• Approaches for investigating VDAC1 interactions with hexokinase, Bcl-2 family proteins, and other binding partners