NDUFA5 Antibody

What is NDUFA5 and what cellular functions does it serve?

NDUFA5 is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) that functions in the transfer of electrons from NADH to the respiratory chain. While it is not directly involved in catalysis, it is essential for proper complex assembly and function. The protein is also known by several other names including Complex I subunit B13, Complex I-13kD-B, NADH-ubiquinone oxidoreductase 13 kDa-B subunit, and CI-13kD-B . NDUFA5 is critical for electron transport and ATP production in the mitochondria, making it a key player in cellular energy metabolism. Dysregulation has been linked to various diseases, including mitochondrial disorders and neurodegenerative conditions .

What molecular characteristics define NDUFA5 protein?

NDUFA5 has a calculated and observed molecular weight of 13 kDa . The full amino acid sequence of human NDUFA5 (NP_004991.1) is: "MAGVLKKTTGLVGLAVCNTPHERLRILYTKILEDVLEEIPKNAAYRKYTEQITNEKLAMVKAEPDVKKLEDQLQGGQLEECVILQAEHELNELARKMREWKLWEPLVEEEPPADQWKWPI" . The protein belongs to the complex I NDUFA5 subunit family and has the UniProt ID Q16718 . The gene encoding NDUFA5 has the NCBI Gene ID 4698 and GenBank accession number BC000813 .

Which species show cross-reactivity with available NDUFA5 antibodies?

Commercial NDUFA5 antibodies show reactivity across multiple species. The Abcam antibody (ab241492) reacts with human and mouse samples . The Assay Genie antibody (CAB3976) exhibits reactivity with human, mouse, and rat samples . Similarly, the Proteintech antibody (16640-1-AP) has been validated to react with human, mouse, and rat samples, with positive Western blot detection demonstrated in HeLa cells, mouse brain tissue, rat brain tissue, Jurkat cells, and mouse liver tissue . These cross-reactivity profiles make these antibodies versatile tools for comparative studies across different model organisms.

What are the recommended applications for NDUFA5 antibodies?

NDUFA5 antibodies are validated for multiple applications:

The Proteintech antibody has demonstrated positive immunohistochemistry (IHC) results in mouse cerebellum tissue and human brain tissue, and positive immunofluorescence (IF/ICC) in HepG2 and HeLa cells .

How should NDUFA5 antibodies be stored to maintain optimal activity?

For optimal maintenance of antibody activity, NDUFA5 antibodies should be stored according to manufacturer specifications. The Proteintech antibody (16640-1-AP) should be stored at -20°C in PBS with 0.02% sodium azide and 50% glycerol pH 7.3. It remains stable for one year after shipment, and aliquoting is unnecessary for -20°C storage . Generally, repeated freeze-thaw cycles should be avoided to prevent protein denaturation and loss of antibody function. For working dilutions, storage at 4°C for short periods (1-2 weeks) is typically acceptable, but longer storage should follow manufacturer recommendations.

How can NDUFA5 antibodies be used to study mitochondrial complex I assembly defects?

NDUFA5 antibodies are valuable tools for studying complex I assembly defects through several approaches. Western blotting with NDUFA5 antibodies can detect accumulated subcomplexes indicative of assembly defects. Research has shown that suppression of certain complex I subunits (like NDUFA11) leads to the accumulation of subcomplexes with estimated molecular masses of 550 and 815 kDa, which can be detected using antibodies against various complex I subunits including NDUFA5 .

Researchers can use NDUFA5 antibodies in conjunction with blue native gel electrophoresis (BN-PAGE) to separate intact complex I from subcomplexes, followed by immunoblotting to assess assembly status. Co-immunoprecipitation with NDUFA5 antibodies can identify interacting proteins in various assembly stages. Additionally, immunofluorescence microscopy using NDUFA5 antibodies can visualize the spatial distribution of the protein within mitochondria and assess whether assembly defects affect this distribution. An integrated approach combining these techniques provides comprehensive insights into complex I assembly defects.

What is the relationship between NDUFA5 expression and mitochondrial diseases?

NDUFA5 expression abnormalities have been implicated in various mitochondrial disorders. As an accessory subunit of complex I, NDUFA5 is critical for proper complex formation and function. Dysregulation of NDUFA5 has been linked to mitochondrial disorders and neurodegenerative conditions .

To study these relationships, researchers can use NDUFA5 antibodies to evaluate protein expression levels in patient samples compared to controls. Quantitative Western blotting can reveal whether NDUFA5 is upregulated, downregulated, or abnormally processed in disease states. Immunohistochemistry in tissues affected by mitochondrial diseases can provide spatial information about NDUFA5 distribution. For functional studies, researchers can assess the impact of NDUFA5 abnormalities on complex I activity, measuring oxygen consumption and ATP production in cellular models. This multi-faceted approach helps establish connections between NDUFA5 expression patterns and mitochondrial disease pathogenesis.

How do mutations in NDUFA5 affect mitochondrial network morphology and what methods detect these effects?

Mutations in complex I subunits, including NDUFA5, can significantly impact mitochondrial network morphology. Research has shown that suppression of expression of complex I subunits can lead to fragmentation of the mitochondrial network that is normally observed in control cells .

To detect and characterize these effects, researchers can employ:

• Live-cell imaging with mitochondria-specific dyes (e.g., MitoTracker) in cells expressing wild-type versus mutant NDUFA5

• Immunofluorescence microscopy using NDUFA5 antibodies alongside mitochondrial markers

• Super-resolution microscopy to precisely analyze mitochondrial morphology changes

• Electron microscopy to examine ultrastructural alterations in mitochondria

• Quantitative image analysis to measure parameters such as mitochondrial length, branching, and network connectivity

These approaches can reveal how NDUFA5 mutations affect not only protein function but also mitochondrial dynamics and morphology, which are increasingly recognized as important factors in mitochondrial disease pathogenesis.

What techniques can assess the impact of NDUFA5 on respiratory complex formation?

Multiple techniques can effectively assess NDUFA5's role in respiratory complex formation:

• Blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by immunoblotting with NDUFA5 antibodies to visualize complex I assembly status

• Sucrose gradient ultracentrifugation to separate respiratory complexes, followed by immunodetection of NDUFA5

• Co-immunoprecipitation using NDUFA5 antibodies to identify interaction partners during complex assembly

• Proximity ligation assays to detect in situ protein-protein interactions between NDUFA5 and other complex I subunits

• CRISPR/Cas9-mediated NDUFA5 knockout or knockdown followed by assessment of complex I formation using antibodies against multiple subunits

Research has demonstrated that perturbations in complex I subunits can lead to the accumulation of subcomplexes, indicating assembly defects . By combining these techniques with functional assays measuring electron transport chain activity, researchers can comprehensively evaluate how NDUFA5 contributes to respiratory complex formation and function.

How can crosslinking studies with NDUFA5 antibodies reveal structural insights?

Crosslinking studies combined with NDUFA5 antibodies offer powerful approaches for gaining structural insights into complex I organization. Researchers can implement the following methodologies:

• Chemical crosslinking with compounds like DSS or formaldehyde to stabilize protein-protein interactions, followed by immunoprecipitation with NDUFA5 antibodies and mass spectrometry analysis to identify proximal proteins

• Photo-activatable crosslinkers incorporated into NDUFA5 or its interaction partners for precisely timed crosslinking upon light activation

• In vivo crosslinking in intact mitochondria followed by NDUFA5 immunoprecipitation to capture physiologically relevant interactions

• Site-specific crosslinking using genetically incorporated unnatural amino acids in NDUFA5, combined with antibody detection to map interaction domains

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) with NDUFA5 antibody pulldown to identify regions involved in protein-protein interactions

These approaches can reveal the spatial organization of NDUFA5 within complex I and identify novel interaction partners, contributing to our understanding of complex I structure and assembly mechanisms.

What are the optimal conditions for Western blot analysis using NDUFA5 antibodies?

Optimal Western blot conditions for NDUFA5 detection require careful consideration of several parameters:

Sample Preparation:

• Use RIPA buffer or other appropriate lysis buffers containing protease inhibitors

• For mitochondrial enrichment, consider subcellular fractionation before analysis

• Typical protein loading: 10-30 μg total protein per lane

Electrophoresis and Transfer:

• Use 12-15% SDS-PAGE gels (appropriate for 13 kDa proteins)

• Transfer to PVDF membranes (preferred over nitrocellulose for small proteins)

• Consider using high-methanol transfer buffers for improved transfer of small proteins

Antibody Incubation:

• Primary antibody dilutions:

  • Proteintech 16640-1-AP: 1:1000-1:6000

  • Assay Genie CAB3976: 1:500-1:2000

• Overnight incubation at 4°C generally yields the best results

• Use 5% non-fat dry milk or BSA in TBST for blocking and antibody dilution

Detection:

• HRP-conjugated secondary antibodies followed by ECL detection work well

• For quantitative analysis, consider fluorescent secondary antibodies

Controls:

• Include positive controls such as HeLa cells, mouse brain tissue, or rat brain tissue

• Consider using NDUFA5 knockdown/knockout samples as negative controls

These conditions should be optimized for each specific laboratory setup and application.

How can immunoprecipitation techniques be optimized for NDUFA5?

Optimizing immunoprecipitation (IP) with NDUFA5 antibodies requires attention to several critical parameters:

Lysis Conditions:

• Use gentle lysis buffers (e.g., 1% NP-40 or 1% Triton X-100) to preserve protein-protein interactions

• Include protease inhibitors and phosphatase inhibitors if post-translational modifications are of interest

• For complex I studies, consider digitonin-based buffers that preserve membrane protein complexes

Antibody Selection and Amount:

• The Abcam antibody (ab241492) has been validated for IP applications

• Typically, 2-6 μg of antibody per 1 mg of total protein is effective

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

IP Protocol:

• For NDUFA5 IP, incubate antibody with lysate overnight at 4°C on a rotator

• Add pre-washed protein A/G beads and incubate for 1-4 hours

• Include multiple gentle washing steps to minimize background

• Elute with either low pH or SDS sample buffer depending on downstream applications

Validation:

• Confirm successful IP by Western blot, loading 20% of IP material

• Include appropriate controls such as IgG-only IP and input lysate

• For co-IP studies, validate interaction partners with specific antibodies

Following these guidelines will enhance the specificity and yield of NDUFA5 immunoprecipitation experiments.

What are the recommended fixation methods for immunohistochemistry with NDUFA5 antibodies?

Effective immunohistochemistry (IHC) for NDUFA5 requires careful consideration of fixation and antigen retrieval methods:

Fixation:

• Formalin fixation (10% neutral buffered formalin) for 24-48 hours is generally effective

• For frozen sections, 4% paraformaldehyde fixation for 10-15 minutes works well

• Avoid overfixation which can mask epitopes, particularly for mitochondrial proteins

Antigen Retrieval:

• Heat-induced epitope retrieval (HIER) is recommended

• Preferred buffer: TE buffer pH 9.0

• Alternative: citrate buffer pH 6.0

• Heating methods: microwave (10-20 minutes), pressure cooker (2-5 minutes), or water bath (20-40 minutes)

Blocking and Antibody Incubation:

• Block with 5-10% normal serum from the species of the secondary antibody

• For Proteintech 16640-1-AP antibody, use 1:50-1:500 dilution for IHC

• Incubate primary antibody overnight at 4°C for optimal signal-to-noise ratio

• Use biotin-free detection systems to minimize background in mitochondria-rich tissues

Controls:

• Positive tissue controls: mouse cerebellum tissue, human brain tissue

• Negative controls: omission of primary antibody and use of tissues known to lack NDUFA5

These parameters should be optimized for specific tissue types and experimental conditions.

How should controls be designed for NDUFA5 immunofluorescence experiments?

Robust controls are essential for reliable NDUFA5 immunofluorescence experiments:

Positive Controls:

• Cell lines with known NDUFA5 expression (e.g., HeLa, HepG2)

• Co-staining with established mitochondrial markers (e.g., TOM20, MitoTracker)

• Overexpression controls using tagged NDUFA5 constructs

Negative Controls:

• Primary antibody omission to assess non-specific binding of secondary antibodies

• Isotype control antibodies to evaluate Fc receptor binding

• NDUFA5 knockdown/knockout cells to confirm antibody specificity

• Pre-absorption of antibody with immunizing peptide when available

Methodological Controls:

• Multiple fixation methods (e.g., paraformaldehyde, methanol) to exclude fixation artifacts

• Serial dilution of primary antibody to determine optimal concentration (starting with recommended 1:50-1:500 for Proteintech antibody)

• Secondary antibody-only controls for each fluorophore used in multi-color experiments

Technical Validation:

• For critical experiments, validate findings with a second NDUFA5 antibody raised against a different epitope

• Confirm immunofluorescence results with an orthogonal method (e.g., Western blotting)

• Include wild-type vs. treated samples in the same experiment to control for technical variables

Implementing these controls ensures confidence in the specificity and reliability of NDUFA5 immunofluorescence results.

What is the optimal approach for studying NDUFA5 in different cell types and tissues?

Studying NDUFA5 across different cell types and tissues requires tailored approaches:

Tissue-Specific Considerations:

• Brain tissue: Requires careful fixation and extended antigen retrieval; mouse cerebellum and human brain have been validated for NDUFA5 IHC

• Muscle tissue: Consider using frozen sections to preserve enzymatic activity for correlation with complex I function

• Highly vascularized tissues: Implement additional blocking steps to reduce background

• Cell lines: HeLa, HepG2, and Jurkat cells have demonstrated detectable NDUFA5 expression

Protocol Adaptations:

• Adjust fixation times based on tissue density and permeability

• Optimize antibody concentration for each tissue/cell type:

  • Start with manufacturer recommendations (e.g., 1:50-1:500 for IHC, 1:1000-1:6000 for WB)

  • Perform titration experiments to determine optimal concentration

• Consider tissue-specific blocking reagents (e.g., mouse-on-mouse blocking for mouse tissues with mouse antibodies)

Multi-Modal Analysis:

• Combine IHC with functional assays specific to the tissue/cell type

• For tissues with high autofluorescence, consider non-fluorescent detection methods

• Use laser capture microdissection followed by immunoblotting for region-specific analysis

• Implement multiplexed immunofluorescence to study NDUFA5 in specific cell populations within heterogeneous tissues

Validation Strategy:

• Always include tissue-matched positive and negative controls

• Validate antibody specificity in each new tissue/cell type

• Consider complementary techniques (e.g., in situ hybridization) to correlate protein with mRNA expression

This systematic approach ensures reliable NDUFA5 detection across diverse biological systems.

Why might Western blots with NDUFA5 antibodies show multiple bands and how should this be interpreted?

Multiple bands on NDUFA5 Western blots can arise from several sources and require careful interpretation:

Potential Causes:

• Post-translational modifications: Phosphorylation, ubiquitination, or other modifications can cause band shifts

• Protein isoforms: Although not widely reported for NDUFA5, alternative splicing could generate different isoforms

• Degradation products: Incomplete protease inhibition during sample preparation can lead to partial degradation

• Complex formation: Incompletely denatured complex I subcomplexes might appear as higher molecular weight bands

• Non-specific binding: The antibody may cross-react with structurally similar proteins

Interpretation Strategies:

• Compare observed band pattern with expected molecular weight (13 kDa for NDUFA5)

• Validate primary band using knockdown/knockout samples or competition with immunizing peptide

• Analyze subcellular fractions (e.g., mitochondrial vs. cytosolic) to determine compartment-specific patterns

• Use different NDUFA5 antibodies targeting distinct epitopes to confirm specific bands

• Consider treating samples with phosphatases or deglycosylation enzymes to identify post-translational modifications

Reporting Guidelines:

• Clearly indicate which band is considered the specific NDUFA5 signal

• Report all consistently observed bands with their approximate molecular weights

• Provide controls demonstrating specificity for the bands being analyzed

• Maintain consistent sample preparation procedures across experiments for comparable results

Proper interpretation of multiple bands requires systematic investigation of these potential causes and appropriate controls.

How can background signals be reduced when using NDUFA5 antibodies for immunofluorescence?

Minimizing background in NDUFA5 immunofluorescence requires addressing several common sources of non-specific signals:

Fixation and Permeabilization Optimization:

• Test multiple fixatives (e.g., 4% PFA, methanol, acetone) to identify optimal preservation with minimal background

• Adjust permeabilization conditions (e.g., 0.1-0.3% Triton X-100 for 5-15 minutes) to balance antibody accessibility with background

• Consider mild permeabilization methods (e.g., digitonin) for selective plasma membrane permeabilization

Blocking Improvements:

• Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

• Test different blocking agents (e.g., 5% normal serum, 3-5% BSA, commercial blocking buffers)

• Include 0.1-0.3% Triton X-100 in blocking solution to reduce hydrophobic interactions

• For tissues with high endogenous biotin, use biotin/avidin blocking kits

Antibody Optimization:

• Perform antibody titration to determine optimal concentration

• Increase antibody incubation time (overnight at 4°C) while decreasing concentration

• Pre-absorb antibodies with acetone powder from non-relevant species tissues

• Use highly cross-adsorbed secondary antibodies to minimize species cross-reactivity

Washing Enhancements:

• Increase washing steps (5-6 washes of 5-10 minutes each)

• Use detergent-containing wash buffers (0.05-0.1% Tween-20 or Triton X-100)

• Perform high-salt washes (up to 500 mM NaCl) to disrupt low-affinity interactions

Fluorescence Considerations:

• Include antifade reagents to reduce autofluorescence

• Use Sudan Black B (0.1-0.3%) treatment to quench lipofuscin autofluorescence in tissues

• Select fluorophores with emission spectra distinct from tissue autofluorescence

• Implement spectral unmixing during image acquisition for samples with significant autofluorescence

These strategies should be systematically tested and optimized for specific experimental conditions.

What are common causes for inconsistent results in NDUFA5 detection across different cell lines?

Inconsistent NDUFA5 detection across cell lines can stem from multiple factors:

Biological Variables:

• Different expression levels of NDUFA5 across cell types and tissues

• Variation in mitochondrial content and activity between cell lines

• Cell type-specific post-translational modifications affecting epitope accessibility

• Metabolic state differences impacting mitochondrial protein expression

• Cell line authentication issues (misidentified or contaminated lines)

Technical Factors:

• Inadequate lysis of mitochondria in cells with robust mitochondrial membranes

• Variation in fixation efficiency across different cell types

• Cell type-specific autofluorescence or background binding patterns

• Antibody lot-to-lot variation affecting specific cell types differently

• Protocol standardization issues (inconsistent handling across cell lines)

Optimization Strategies:

• Adjust lysis conditions for each cell type (consider stronger detergents for resistant cell lines)

• Standardize cell culture conditions (confluence, passage number, media composition)

• Normalize to mitochondrial content markers (e.g., VDAC, COX IV) rather than conventional housekeeping genes

• Validate antibody performance in each new cell line with positive and negative controls

• Implement subcellular fractionation to enrich mitochondrial proteins before analysis

Data Integration Approach:

• Use multiple detection methods (WB, IF, flow cytometry) to cross-validate findings

• Correlate protein detection with functional assays of complex I activity

• Quantify relative rather than absolute expression levels when comparing across cell lines

• Consider targeted proteomics approaches for absolute quantification

• Validate key findings with orthogonal approaches (e.g., mRNA expression, activity assays)

This comprehensive approach addresses both biological and technical sources of variability in NDUFA5 detection.

How should conflicting NDUFA5 expression data be reconciled between RNA and protein analysis?

Discrepancies between NDUFA5 RNA and protein levels require systematic investigation and reconciliation:

Common Causes of RNA-Protein Discordance:

• Post-transcriptional regulation (miRNAs, RNA-binding proteins)

• Differences in half-life between mRNA (typically hours) and proteins (typically days)

• Translational efficiency variations across cell types or conditions

• Protein degradation pathways specifically targeting NDUFA5

• Technical limitations in detection methods for either RNA or protein

Analytical Approaches:

• Temporal analysis: Measure both RNA and protein at multiple time points to capture dynamic relationships

• Subcellular fractionation: Confirm proper localization of NDUFA5 protein to mitochondria

• Polysome profiling: Assess translational efficiency of NDUFA5 mRNA

• Pulse-chase experiments: Determine NDUFA5 protein half-life across conditions

• Proteasome/autophagy inhibitors: Test if protein degradation explains low protein despite high mRNA

Integration Strategies:

• Normalize RNA and protein data to appropriate internal controls

• Consider ratios or fold changes rather than absolute values when comparing across methods

• Validate key findings using multiple antibodies and RNA detection methods

• Correlate both RNA and protein data with functional assays (e.g., complex I activity)

• Implement targeted experimental designs to test specific hypotheses about the discrepancy

Reporting Recommendations:

• Clearly acknowledge discrepancies rather than selectively reporting concordant data

• Present both RNA and protein data together with appropriate statistical analysis

• Discuss biological implications of discordance rather than dismissing it as technical artifact

• Consider the discrepancy itself as a finding that may reveal important regulatory mechanisms

• Propose follow-up studies specifically designed to address the molecular basis of discordance

This systematic approach transforms apparent contradictions into opportunities for deeper biological insights.

What approaches can resolve non-specific binding issues with NDUFA5 antibodies?

Resolving non-specific binding requires a multi-faceted approach:

Antibody Validation Strategies:

• Test multiple NDUFA5 antibodies targeting different epitopes

• Validate with NDUFA5 knockout/knockdown controls

• Perform peptide competition assays with the immunizing peptide

• Check for cross-reactivity with other complex I subunits with similar structures

Protocol Optimization:

• Increase blocking stringency (longer time, higher concentration, different blocking agents)

• Add carrier proteins (e.g., 0.1-0.5% BSA) to antibody dilution buffers

• Include mild detergents (0.05-0.1% Tween-20) in all buffers

• Perform high-salt washes (300-500 mM NaCl) to disrupt low-affinity interactions

• Test gradient dilutions of primary antibody to find optimal signal-to-noise ratio

Sample Preparation Improvements:

• Ensure complete denaturation for Western blotting (heat samples at 95°C for 5 minutes in SDS buffer)

• Use fresh samples and avoid repeated freeze-thaw cycles

• Include additional protease inhibitors to prevent degradation products

• For difficult tissues, consider antigen retrieval optimization

• Test different fixation methods for immunohistochemistry/immunofluorescence

Advanced Purification Techniques:

• Pre-absorb antibodies against tissues or lysates from knockout models

• Use immunoaffinity purification to isolate the specific antibody fraction

• Implement dual-labeling strategies to distinguish specific from non-specific signals

• Consider proximity ligation assays that require dual epitope recognition

• Use gentle crosslinking to stabilize specific interactions before harsh lysis

Signal Detection Optimization:

• Reduce exposure time/gain to minimize background amplification

• Implement spectral unmixing for autofluorescent samples

• Use secondary antibodies with minimal cross-reactivity to the species being studied

• Consider signal amplification methods only after optimizing primary detection

• For quantitative applications, establish signal linearity range and stay within it

Implementing these strategies systematically can significantly improve NDUFA5 antibody specificity across applications.