NDUFV2 Antibody

What is NDUFV2 and what cellular functions does it perform?

NDUFV2 functions as a core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I), catalyzing electron transfer from NADH through the respiratory chain with ubiquinone as the electron acceptor. The protein belongs to the complex I 24 kDa subunit family and constitutes part of the peripheral arm of the enzyme complex. Within this structure, electrons from NADH are accepted by flavin mononucleotide (FMN) and passed along iron-sulfur clusters by electron tunneling to the final acceptor ubiquinone. NDUFV2 itself contains one iron-sulfur cluster and is considered essential for catalytic function of Complex I. Notably, NDUFV2 has been identified as a potential genetic risk factor for Parkinson's Disease, with mutations potentially causing Complex I deficiency associated with this neurodegenerative condition .

What applications are NDUFV2 antibodies validated for?

NDUFV2 antibodies have been validated for multiple research applications, with performance characteristics varying by antibody clone and format:

For optimal results, researchers should titrate antibodies for their specific experimental system to determine optimal working concentrations .

What species reactivity do NDUFV2 antibodies display?

NDUFV2 antibodies demonstrate variable cross-reactivity among species depending on the antibody clone and manufacturing process. Comprehensive testing has confirmed the following reactivity patterns:

When working with species not listed in validation data, researchers should perform preliminary validation experiments with appropriate positive controls from the target species before proceeding with full experiments .

What positive control samples are recommended for NDUFV2 antibody validation?

Successful validation of NDUFV2 antibodies requires appropriate positive control samples. Based on published validation data, the following tissues and cell lines are recommended:

These samples consistently show robust NDUFV2 expression and serve as reliable controls for antibody performance assessment. For knockout validation approaches, NDUFV2 knockout cell lines (e.g., HeLa NDUFV2 KO) have successfully demonstrated antibody specificity when compared with wild-type controls .

What are the optimal storage conditions for NDUFV2 antibodies?

Proper storage is critical for maintaining NDUFV2 antibody performance over time. Most commercial NDUFV2 antibodies require:

• Storage at -20°C for long-term stability

• Storage buffer typically containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Stability for approximately one year after shipment when stored properly

• No requirement for aliquoting when stored at -20°C for many formulations

For opened/reconstituted antibody products, manufacturers recommend using within one month. After thawing, avoid repeated freeze-thaw cycles which can degrade antibody performance. Some preparations (20μL sizes) may contain 0.1% BSA as a stabilizer. Always refer to product-specific storage instructions, as formulations may vary between manufacturers .

How should Western blot protocols be optimized for detecting NDUFV2?

Optimizing Western blot protocols for NDUFV2 detection requires attention to several technical parameters:

Sample Preparation Considerations:

• Effective mitochondrial protein extraction is essential as NDUFV2 localizes to mitochondria

• Use of protease inhibitors is critical to prevent degradation

• Gentle lysis methods help preserve native protein structure

Protocol Optimization Parameters:

• Expected molecular weight range: 24-27 kDa (observed), 27 kDa (calculated)

• Recommended antibody dilutions vary significantly between antibody clones (1:5000-1:50000)

• Standard SDS-PAGE conditions with 10-12% gels provide optimal resolution

• Transfer conditions: lower current for longer time enhances transfer of this mitochondrial protein

• Blocking: 5% non-fat milk in TBST for 1-2 hours at room temperature

• Primary antibody incubation: overnight at 4°C for optimal signal-to-noise ratio

Validation Strategy:
Compare results with known positive controls (heart tissue, brain tissue) and, ideally, NDUFV2 knockout samples to confirm band specificity. The specific antibody clone EPR15351(B) has been validated using knockout cell lines (HeLa NDUFV2 KO), confirming its specificity for the intended target .

What antigen retrieval methods are optimal for NDUFV2 immunohistochemistry?

Effective antigen retrieval is crucial for successful NDUFV2 immunohistochemistry. Based on published protocols and manufacturer recommendations:

Primary Recommended Method:

• TE buffer at pH 9.0 is the preferred antigen retrieval solution for most NDUFV2 antibodies

• Heat-induced epitope retrieval (HIER) using a pressure cooker or microwave heating system

• Heating time of 15-20 minutes followed by gradual cooling to room temperature

Alternative Method:

• Citrate buffer at pH 6.0 may also be effective for certain antibody clones

• This method may be preferred when using multiplexing approaches with other antibodies

Tissue-Specific Considerations:

• For heart tissue, which shows strong NDUFV2 expression, milder retrieval conditions may be sufficient

• For brain tissue, more extensive retrieval may be necessary to penetrate the tissue effectively

• Formalin-fixed paraffin-embedded (FFPE) tissues require more rigorous retrieval than frozen sections

Following retrieval, recommended dilutions for IHC applications range from 1:500-1:2000, with specific optimization required for each tissue type and fixation method .

What are the key differences between monoclonal and polyclonal NDUFV2 antibodies for research applications?

Monoclonal and polyclonal NDUFV2 antibodies offer distinct advantages and limitations that impact experimental design choices:

For critical experiments, parallel validation using both antibody types can provide complementary data and increase confidence in results. Recombinant monoclonal antibodies like EPR15351(B) offer the specificity advantages of monoclonals with improved consistency and reduced batch variation .

How can NDUFV2 antibodies be employed to investigate mitochondrial dysfunction in neurodegenerative disorders?

NDUFV2 antibodies serve as valuable tools for investigating mitochondrial dysfunction in neurodegenerative conditions, particularly since NDUFV2 has been identified as a genetic risk factor for Parkinson's Disease:

Research Applications:

• Quantification of NDUFV2 protein levels in affected brain regions using Western blot and IHC

• Analysis of NDUFV2 localization and distribution patterns using immunofluorescence microscopy

• Assessment of Complex I assembly and integrity using co-immunoprecipitation with NDUFV2 antibodies

• Evaluation of post-translational modifications affecting NDUFV2 function

• Comparison of NDUFV2 levels between patient and control samples

Experimental Approaches:

• Brain tissue section analysis using IHC with anti-NDUFV2 antibodies to identify region-specific changes

• Patient-derived cellular models (fibroblasts, iPSC-derived neurons) probed with NDUFV2 antibodies

• Co-labeling with markers of oxidative stress to correlate with NDUFV2 alterations

• Biochemical isolation of mitochondrial fractions followed by NDUFV2 immunoblotting

• Functional assays of Complex I activity correlated with NDUFV2 protein levels

Published studies have successfully employed these approaches in PD research, demonstrating altered NDUFV2 levels or localization in disease models. Researchers should consider background strain differences when working with mouse models and tissue-specific optimization of antibody protocols .

What validation strategies confirm specificity of NDUFV2 antibodies?

Genetic Validation:

• Testing with NDUFV2 knockout cell lines (e.g., HeLa NDUFV2 KO ab265619)

• RNA interference (siRNA/shRNA) to create NDUFV2 knockdown models

• Overexpression systems with tagged NDUFV2 constructs

Analytical Validation:

• Western blot analysis confirming single band at expected molecular weight (24-27 kDa)

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Competitive blocking with immunizing peptide/antigen

• Cross-validation using multiple antibodies targeting different NDUFV2 epitopes

Application-Specific Validation:

• For IHC/IF: Comparison to mRNA expression patterns (in situ hybridization)

• For flow cytometry: Parallel validation with known mitochondrial markers

• Positive controls using tissues with known high NDUFV2 expression (heart, brain)

Commercial antibody ab183715 (EPR15351(B)) demonstrated exemplary validation through complete signal loss in NDUFV2 knockout cell lysates compared to wild-type controls, establishing definitive specificity for the intended target .

What technical challenges arise when working with NDUFV2 antibodies and how can they be addressed?

Researchers working with NDUFV2 antibodies may encounter several technical challenges that require specific troubleshooting approaches:

Challenge 1: Weak or Absent Signal

• Potential Causes: Insufficient antigen retrieval, suboptimal antibody concentration, degraded antibody

• Solutions:

  • Optimize antigen retrieval using recommended TE buffer pH 9.0

  • Test multiple antibody dilutions in a titration experiment

  • Confirm antibody viability with positive control samples

  • Extend primary antibody incubation time (overnight at 4°C)

Challenge 2: High Background

• Potential Causes: Excessive antibody concentration, insufficient blocking, non-specific binding

• Solutions:

  • Increase dilution factor (especially for monoclonal antibodies which can be used at 1:50000)

  • Extend blocking step (5% BSA or milk for 2 hours)

  • Include 0.1-0.3% Triton X-100 in wash buffers

  • For IHC, treat with hydrogen peroxide to block endogenous peroxidases

Challenge 3: Multiple Bands in Western Blot

• Potential Causes: Protein degradation, post-translational modifications, non-specific binding

• Solutions:

  • Use fresh samples with complete protease inhibitor cocktails

  • Compare with knockout/knockdown controls to identify specific band

  • Optimize washing conditions (longer washes, higher detergent concentration)

  • Consider using monoclonal antibodies with higher specificity

Challenge 4: Inconsistent Results Between Applications

• Potential Causes: Epitope accessibility differences, protocol-specific requirements

• Solutions:

  • Select application-validated antibodies (not all NDUFV2 antibodies work equally in all applications)

  • Adjust protocols for specific applications following manufacturer recommendations

  • Consider using different antibody clones for different applications

How should researchers design multiplexing experiments incorporating NDUFV2 antibodies?

Multiplexing experiments that incorporate NDUFV2 antibodies with other markers require careful design considerations:

Antibody Selection Parameters:

• Host species compatibility to prevent cross-reactivity between secondary antibodies

• Ensure primary antibodies are raised in different host species (e.g., rabbit anti-NDUFV2 paired with mouse anti-mitochondrial markers)

• Fluorophore selection with non-overlapping emission spectra

• Consider using directly conjugated antibodies when possible

Experimental Design Strategy:

• Begin with sequential staining protocols to establish individual antibody performance

• For IHC multiplexing, test antigen retrieval conditions compatible with all targets

• When using anti-NDUFV2 with other mitochondrial markers, carefully control for potential epitope competition

• Include single-stain controls for spectral compensation and antibody performance validation

NDUFV2-Specific Considerations:

• As a mitochondrial protein, co-staining with mitochondrial membrane markers provides contextual localization

• For neurodegenerative disease research, pair NDUFV2 staining with neuronal markers and/or pathological hallmarks

• When investigating oxidative stress, combine with markers of ROS damage

• Consider subcellular fractionation approach before immunostaining to enhance specificity

Successful multiplexing has been demonstrated using rabbit monoclonal NDUFV2 antibodies (e.g., EPR15351(B)) paired with mouse antibodies against other targets, particularly in studies examining mitochondrial complex integrity and localization .

What quantitative approaches accurately measure NDUFV2 protein levels?

Accurate quantification of NDUFV2 protein levels requires rigorous methodological approaches:

Western Blot Quantification:

• Normalization to multiple loading controls is essential (both total protein stains and housekeeping proteins)

• For mitochondrial content normalization, include parallel blots for mitochondrial markers (e.g., VDAC, COX4)

• Densitometric analysis should utilize the linear range of detection

• Multiple biological and technical replicates (minimum n=3) are necessary for statistical validity

ELISA-Based Quantification:

• Commercial NDUFV2 ELISA kits (e.g., RK11534) employ sandwich enzyme immunoassay technique

• Standard curve preparation is critical for accurate concentration determination

• Sample preparation protocols must be consistent across experimental groups

• Appropriate dilution of samples ensures readings within the linear range of detection

Mass Spectrometry Approaches:

• Targeted proteomics using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM)

• Incorporation of isotope-labeled internal standards for absolute quantification

• Integration with immunoprecipitation can enhance specificity for NDUFV2 analysis

• Enables simultaneous quantification of post-translational modifications

Considerations for Sample Types:

• Tissue homogenates: Normalization to total protein is essential

• Cell culture: Account for variations in mitochondrial content

• Body fluids: Pre-concentration may be required due to low abundance

The sandwich ELISA approach offers particular advantages for quantification, utilizing pre-coated microplates with antibodies specific for Human NDUFV2, followed by detection antibody binding and enzymatic color development proportional to NDUFV2 concentration .