NDUFAF3 Antibody, FITC conjugated

What is NDUFAF3 and why is it significant in mitochondrial research?

NDUFAF3 (also known as C3ORF60) is a genuine mitochondrial complex I assembly protein that interacts with complex I subunits during the assembly process. It plays a crucial role in the biogenesis of the Q-, N-, and P-modules of complex I. Research has demonstrated that NDUFAF3 tightly interacts with NDUFAF4 (C6ORF66), another protein implicated in complex I deficiency . Gene conservation analysis has linked NDUFAF3 to bacterial membrane insertion gene cluster SecF/SecD/YajC and to C8ORF38, which is also implicated in complex I deficiency . Mutations in the NDUFAF3 gene have been identified in patients with isolated complex I deficiency, highlighting its clinical significance in mitochondrial disorders . Understanding NDUFAF3 function is essential for elucidating the molecular mechanisms underlying complex I assembly defects.

What experimental applications is NDUFAF3 Antibody, FITC conjugated suitable for?

• Immunocytochemistry - For subcellular localization studies of NDUFAF3

• Flow cytometry - Leveraging the FITC conjugation for direct detection

• Confocal microscopy - For co-localization studies with other mitochondrial proteins

When designing experiments, consider that this antibody is a polyclonal antibody raised in rabbit against recombinant Human NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 3 protein (2-84AA) . The polyclonal nature may provide broader epitope recognition but potentially higher background compared to monoclonal alternatives.

How should researchers validate the specificity of NDUFAF3 Antibody, FITC conjugated?

Validation of NDUFAF3 Antibody, FITC conjugated should follow multiple complementary approaches:

• Positive controls: Use cell lines known to express NDUFAF3 (e.g., HEK293 cells with inducible NDUFAF3-GFP as described in research protocols)

• Negative controls: Include:

  • Secondary antibody-only controls

  • Cells with NDUFAF3 knockdown using validated siRNA (e.g., sequences targeting NDUFAF3: 5′-AUGUAAGUGAAGUCCCUCC dTdT-3′ or 5′-AGGAAGUUGAAGGUGGCAC dTdT-3′)

• Western blot verification: Confirm a single band at the expected molecular weight of NDUFAF3 (~9.3 kDa)

• Subcellular co-localization: Verify mitochondrial localization using mitochondrial markers such as Mitotracker Red

What are the optimal storage and handling conditions for NDUFAF3 Antibody, FITC conjugated?

The NDUFAF3 Antibody, FITC conjugated should be stored according to the manufacturer's specifications:

To maintain fluorescence activity of the FITC conjugate, protect the antibody from light during storage and handling. When working with the antibody, aliquot into smaller volumes upon first thaw to minimize freeze-thaw cycles that can degrade both antibody function and fluorescence intensity .

How can NDUFAF3 Antibody, FITC conjugated be used to investigate mitochondrial complex I assembly defects?

NDUFAF3 Antibody, FITC conjugated can be strategically employed to study complex I assembly defects through several sophisticated approaches:

• Blue-Native PAGE followed by immunoblotting: This technique allows visualization of different assembly intermediates. After BN-PAGE (5%-15% gradient), transfer proteins to PROTAN nitrocellulose membrane and probe with NDUFAF3 Antibody to identify assembly complexes containing NDUFAF3 .

• Two-dimensional analysis: Combine BN-PAGE with second-dimension SDS-PAGE (10%) to separate individual proteins from complexes. This reveals the integration of NDUFAF3 into assembly intermediates and its interactions with other subunits .

• Comparative analysis in patient samples: Compare complex I assembly patterns between control fibroblasts and fibroblasts from patients with NDUFAF3 mutations using the following methodology:

  • Isolate mitochondria using differential centrifugation

  • Measure complex I activity using NADH-ferricyanide reductase, rotenone-sensitive NADH-ubiquinone reductase, and rotenone-sensitive NADH-cytochrome c reductase assays

  • Normalize activities to citrate synthase or cytochrome c oxidase

  • Correlate with assembly pattern visualization

The antibody helps identify where in the assembly process defects occur, particularly in the Q-, N-, and P-modules of complex I, which are impaired when NDUFAF3 is disrupted .

What methodology should be used to study the interaction between NDUFAF3 and NDUFAF4 using NDUFAF3 Antibody, FITC conjugated?

To investigate the NDUFAF3-NDUFAF4 interaction, implement the following methodological approach:

• Co-immunoprecipitation with dual detection:

  • Perform IP with anti-NDUFAF4 antibody

  • Detect NDUFAF3 using FITC-conjugated NDUFAF3 antibody

  • The FITC conjugation allows direct visualization without secondary antibody

• Tandem Affinity Purification coupled with FT-MS:

  • Generate NDUFAF3-TAP and NDUFAF4-TAP constructs

  • Perform TAP according to the InterPlay Mammalian TAP System protocol

  • Elute proteins in 50 mM NH₄HCO₃ (pH 8.0) at 95°C for 5 minutes

  • Analyze by FT-MS using a nano-HPLC Agilent 1100 nanoflow system connected to an LTQ-FT mass spectrometer

  • Identify interacting proteins using Mascot algorithm and MSQuant for generating unique first-ranked peptide lists

• Proximity ligation assay:

  • Use FITC-conjugated NDUFAF3 antibody with unconjugated NDUFAF4 antibody

  • Employ complementary proximity probes to detect close interaction (<40 nm)

  • Quantify interaction signals relative to appropriate controls

Research has demonstrated that NDUFAF3 and NDUFAF4 tightly interact and work together in complex I assembly. Notably, forced expression of NDUFAF4 can rescue biogenesis defects in the Q-module and some aspects of the P-module when NDUFAF3 is disrupted .

How can researchers optimize protocols for using NDUFAF3 Antibody, FITC conjugated in confocal microscopy?

For optimal confocal microscopy with NDUFAF3 Antibody, FITC conjugated, implement the following protocol refinements:

• Sample preparation:

  • Culture cells in appropriate vessels (e.g., Wilco dishes) suitable for high-resolution imaging

  • For co-localization studies, combine with mitochondrial markers (e.g., incubate with 1 μM Mitotracker Red for 15 min at 37°C)

  • For nuclear counterstaining, use 10 μM Hoechst 3342 for 30 min at 37°C

• Imaging parameters:

  • Replace culture medium with colorless HEPES-Tris solution (132 mM NaCl, 4.2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 5.5 mM D-glucose, 10 mM HEPES, pH 7.4) to reduce background

  • Acquire images at a rate of 10 Hz using a 63× oil-immersion objective (N.A. 1.4)

  • Use appropriate filter sets for FITC (excitation ~495 nm, emission ~519 nm)

• Controls and validation:

  • Include untransfected cells as negative controls

  • Use cells expressing NDUFAF3-GFP as positive controls for localization pattern comparison

  • For co-localization studies, calculate Pearson's correlation coefficient to quantify the degree of co-localization

• Considerations for photobleaching:

  • Minimize exposure time to reduce FITC photobleaching

  • Consider time-lapse imaging with reduced laser power for dynamic studies

  • For FRAP studies, determine optimal bleaching parameters empirically

What are the experimental considerations for studying NDUFAF3's role in complex I module assembly?

When investigating NDUFAF3's role in complex I module assembly, researchers should consider the following methodological approaches:

• Module-specific analysis:

  • Based on research findings, NDUFAF3 disruption impairs biogenesis of the Q-, N-, and P-modules of complex I

  • Design experiments to individually assess each module's assembly

• Integration of key subunits:

  • Focus on NDUFS3 integration into the Q-module

  • Examine NDUFS5 integration into the P-module

  • Both are compromised when NDUFAF3 is disrupted

• TIMMDC1 stability assessment:

  • Monitor TIMMDC1 (another complex I assembly factor) stability

  • TIMMDC1 destabilization in assembly intermediates occurs with NDUFAF3 disruption

• Rescue experiments:

  • Test forced expression of NDUFAF4 to rescue biogenesis defects

  • Research shows NDUFAF4 overexpression can rescue defects in the Q-module and some aspects of the P-module when NDUFAF3 is disrupted

• Experimental system selection:

  • Consider Drosophila genetic models for in vivo studies

  • Use human cell lines (e.g., HEK293) for biochemical analyses

  • Patient-derived fibroblasts provide disease-relevant context

How can researchers employ NDUFAF3 Antibody, FITC conjugated in multi-parametric flow cytometry studies?

For multi-parametric flow cytometry using NDUFAF3 Antibody, FITC conjugated, implement the following methodological approach:

• Panel design considerations:

  • FITC emission spectrum: 519 nm (green channel)

  • Compatible fluorophores with minimal spectral overlap:

    • PE (575 nm) for mitochondrial markers

    • APC (660 nm) for cell surface markers

    • BV421 (421 nm) for additional intracellular targets

• Sample preparation protocol:

  • Fix cells with 2% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% saponin buffer to access intracellular antigens

  • Block with 3% BSA to reduce non-specific binding

  • Apply NDUFAF3 Antibody, FITC conjugated at empirically determined optimal concentration

• Compensation strategy:

  • Prepare single-color controls for each fluorophore

  • Include fluorescence-minus-one (FMO) controls to set gates accurately

  • Use automated compensation matrices with manual adjustment

• Data analysis approach:

  • Gate on viable single cells before analyzing NDUFAF3 expression

  • Correlate NDUFAF3 levels with mitochondrial mass markers

  • Consider median fluorescence intensity (MFI) for quantitative comparisons

• Potential applications:

  • Correlation of NDUFAF3 expression with mitochondrial membrane potential

  • Cell cycle-dependent changes in NDUFAF3 expression

  • Effects of metabolic stress on NDUFAF3 levels

How can NDUFAF3 Antibody, FITC conjugated be utilized in patient-derived samples to study mitochondrial disorders?

For clinical research applications using patient-derived samples, consider these methodological approaches:

• Diagnostic workflow integration:

  • Patient fibroblast analysis workflow:

    • Isolate mitochondria from patient fibroblasts

    • Measure complex I enzyme activity (NADH-ferricyanide reductase, rotenone-sensitive NADH-ubiquinone reductase, and NADH-cytochrome c reductase assays)

    • Normalize to citrate synthase or cytochrome c oxidase

    • Correlate enzymatic defects with NDUFAF3 expression/localization

• Genotype-phenotype correlation studies:

  • In patients with NDUFAF3 mutations, assess:

    • NDUFAF3 protein expression levels

    • NDUFAF3 subcellular localization

    • Complex I assembly status

    • Correlation with clinical severity

• Therapeutic response monitoring:

  • Baseline assessment of NDUFAF3 expression and complex I assembly

  • Monitor changes following experimental therapeutics

  • Potential for NDUFAF4 overexpression as a therapeutic approach for certain NDUFAF3 mutations

• Biochemical analysis of patient samples:

  • BN-PAGE analysis to detect assembly intermediates

  • Two-dimensional SDS-PAGE to assess subunit incorporation

  • Western blotting with antibodies against multiple complex I subunits (NDUFS3, ND1, NDUFS2, NDUFA2, NDUFA5, NDUFA9)

What experimental design should be implemented to investigate the cooperative function of NDUFAF3 and NDUFAF4 in complex I assembly?

To study the cooperative function of NDUFAF3 and NDUFAF4, implement the following comprehensive experimental design:

• Sequential knockdown experiments:

  • Use validated siRNA sequences:

    • NDUFAF3 #1: 5′-AUGUAAGUGAAGUCCCUCC dTdT-3′

    • NDUFAF3 #2: 5′-AGGAAGUUGAAGGUGGCAC dTdT-3′

    • NDUFAF4 #1: 5′-UGGAUAGAGACUAAUCUGC dTdT-3′

    • NDUFAF4 #2: 5′-AUCUUUGGAAUCAACAUAC dTdT-3′

  • Transfect cells twice at 48-hour intervals for optimal knockdown

  • Compare individual and double knockdowns to assess synergistic effects

• Rescue experiments:

  • Generate expression constructs:

    • NDUFAF3-GFP

    • NDUFAF4-GFP

    • NDUFS3-GFP (as a control for complex I assembly)

  • Assess rescue of complex I assembly in knockdown backgrounds

  • Test forced expression of NDUFAF4 to rescue NDUFAF3 deficiency

• Interaction mapping:

  • Perform domain mapping through truncation constructs

  • Use tandem affinity purification to identify interaction domains

  • Confirm through site-directed mutagenesis of key residues

• Assembly intermediate characterization:

  • Fractionate mitochondria to separate peripheral and integral membrane proteins

  • Analyze assembly intermediates by BN-PAGE followed by in-gel activity assays

  • Perform second-dimension SDS-PAGE to resolve individual components

Research has shown that NDUFAF4 overexpression can rescue biogenesis defects in both the Q-module and some aspects of the P-module when NDUFAF3 is disrupted, suggesting a compensatory mechanism that could be therapeutically relevant .

What are common technical challenges when using NDUFAF3 Antibody, FITC conjugated and how can they be addressed?

When working with NDUFAF3 Antibody, FITC conjugated, researchers may encounter these technical challenges with corresponding solutions:

How can researchers optimize NDUFAF3 Antibody, FITC conjugated for dual immunofluorescence applications?

For dual immunofluorescence applications with NDUFAF3 Antibody, FITC conjugated, implement these optimization strategies:

• Compatible secondary target selection:

  • Choose targets with distinct subcellular localization from NDUFAF3

  • For mitochondrial co-localization studies, select markers from different submitochondrial compartments

  • Consider antibodies raised in species other than rabbit to avoid cross-reactivity

• Fluorophore selection:

  • Pair FITC (excitation: 495 nm, emission: 519 nm) with fluorophores having minimal spectral overlap:

    • TRITC/Cy3 (excitation: 550 nm, emission: 570 nm)

    • Alexa Fluor 647 (excitation: 650 nm, emission: 668 nm)

• Sequential staining protocol:

  • Apply unconjugated primary antibody first

  • Add corresponding secondary antibody

  • Block with normal rabbit serum to prevent cross-reactivity

  • Apply NDUFAF3 Antibody, FITC conjugated last

• Control experiments:

  • Single-stained controls to assess bleed-through

  • Secondary-only controls to evaluate background

  • Absorption controls with excess unlabeled antibody

• Image acquisition strategy:

  • Capture images sequentially rather than simultaneously

  • Begin with longer wavelengths to minimize photobleaching of FITC

  • Adjust detector gain separately for each channel