ACAD9 Antibody, FITC conjugated

What are the optimal storage and handling conditions for ACAD9 antibody with FITC conjugation?

For optimal performance of FITC-conjugated ACAD9 antibodies, store the antibody at 4°C in the dark for short-term use (up to one month) or at -20°C for long-term storage (aliquoted to avoid freeze-thaw cycles). FITC conjugates are particularly sensitive to light exposure, which can lead to photobleaching and reduced signal intensity. When handling the antibody, minimize exposure to room temperature and bright light. Prior to use, centrifuge the antibody vial to collect the solution at the bottom. For immunofluorescence applications, dilute in appropriate buffers containing 1% BSA or normal serum from the same species as the secondary antibody to reduce background staining. Based on experimental protocols for other antibodies in ACAD research, typical working dilutions range from 1:1000 to 1:3000, but optimal concentrations should be determined empirically for each application .

What sample preparation techniques are recommended for ACAD9 antibody detection?

Sample preparation depends on the experimental application. For immunoblotting, protocols using 50 μg of total protein from human fibroblasts or liver extracts and 150 μg from muscle lysates have been successfully employed with ACAD9 antibodies . Samples should be separated on 12% SDS polyacrylamide gels and transferred to nitrocellulose membranes. For tissue sections or cultured cells intended for immunofluorescence using FITC-conjugated antibodies, fixation with 4% paraformaldehyde followed by permeabilization with 0.1-0.5% Triton X-100 is recommended. Blocking with 5-10% normal serum or BSA helps reduce background signals. For flow cytometry applications, cells should be fixed with 2-4% paraformaldehyde and permeabilized if detecting intracellular ACAD9, as it is primarily located in mitochondria .

What is the relationship between ACAD9 and other acyl-CoA dehydrogenases?

ACAD9 shares significant homology with other members of the acyl-CoA dehydrogenase family, particularly VLCAD (Very Long-Chain Acyl-CoA Dehydrogenase), with which it shares 46.4% sequence identity and 77.6% sequence similarity . Despite this similarity, ACAD9 exhibits unique structural and functional features. Unlike VLCAD, ACAD9 has a weaker FAD-binding affinity, with purified wild-type ACAD9 containing only about 70% FAD . Additionally, ACAD9's dehydrogenation activity (131 units) is only about 18% of VLCAD's activity (995 units) under the same conditions, even when supplemented with exogenous FAD . Most notably, ACAD9 is the only ACAD family member capable of binding ECSIT and assisting in complex I assembly, a function not shared by VLCAD . These differences are important to consider when designing experiments with ACAD9 antibodies to ensure specificity and avoid cross-reactivity with other ACAD family proteins.

What controls should be included when using FITC-conjugated ACAD9 antibodies?

When designing experiments with FITC-conjugated ACAD9 antibodies, several controls are essential for result validation:

• Negative controls:

  • Isotype control: A FITC-conjugated antibody of the same isotype but irrelevant specificity

  • No primary antibody control: Incubation with secondary reagents only

  • Blocking peptide competition: Pre-incubation of the antibody with purified ACAD9 protein at a 1:1 molar ratio, similar to the immunocompetition assays described in the literature

• Positive controls:

  • Known ACAD9-expressing cells or tissues (fibroblasts, liver, muscle)

  • Recombinant ACAD9 protein expression systems

• Specificity controls:

  • ACAD9 knockout or knockdown cells, such as those from the mouse models described in research

  • Comparison with non-conjugated ACAD9 antibody detection methods

• Technical controls:

  • Autofluorescence control: Unstained samples to assess natural fluorescence

  • Single-color controls for compensation when performing multicolor flow cytometry

How can I distinguish between ACAD9's dual functions in experimental settings?

Distinguishing between ACAD9's roles in fatty acid β-oxidation versus complex I assembly requires specific experimental approaches:

For fatty acid β-oxidation function:

• Measure ACAD activity directly using specific substrates such as palmitoyl-CoA or C16:0-CoA, as well as dimethyl C7-CoA as described in published protocols

• Quantify FAD binding using spectrophotometric methods to assess the cofactor relationship, noting that ACAD9 shows approximately 70% FAD occupancy compared to VLCAD

• Monitor dehydrogenation activity with and without exogenous FAD supplementation

For complex I assembly function:

• Assess interactions with ECSIT and NDUFAF1 using co-immunoprecipitation, as these interactions are specific to ACAD9's assembly role

• Use proximity ligation assays with dual antibody labeling to visualize ACAD9-ECSIT interactions in situ

• Evaluate complex I assembly states in the presence/absence of ACAD9 using blue native gel electrophoresis

The critical experiment demonstrating the mutually exclusive nature of these functions comes from evidence that when ECSIT interacts with ACAD9, the flavoenzyme loses its FAD cofactor and consequently loses its FAO activity, demonstrating that the two roles are not compatible . This can be visualized using FITC-conjugated ACAD9 antibodies in combination with FAD autofluorescence detection or ECSIT labeling with another fluorophore.

What techniques can be used to study the ACAD9-ECSIT-NDUFAF1 complex formation?

The MCIA (Mitochondrial Complex I Assembly) complex formed by ACAD9, ECSIT, and NDUFAF1 can be studied using various approaches:

• Co-immunoprecipitation: Using ACAD9 antibodies to pull down the complex and identify binding partners through western blot. Research has shown that while the binary complex of ACAD9 with ECSIT is unstable and aggregates easily, the ternary complex of ACAD9-ECSIT-NDUFAF1 is soluble and extremely stable .

• Fluorescence microscopy: FITC-conjugated ACAD9 antibodies can be used alongside differently labeled antibodies against ECSIT and NDUFAF1 to visualize co-localization. ECSIT binds to the carboxy-terminal half of ACAD9, while NDUFAF1 binds to the amino-terminal half of ECSIT .

• FRET (Förster Resonance Energy Transfer): Since FITC can serve as a donor fluorophore, it can be paired with an acceptor fluorophore on antibodies against ECSIT or NDUFAF1 to measure protein-protein interactions through energy transfer.

• Structural analysis approaches: Research has employed molecular modeling and SAXS (Small-Angle X-ray Scattering) studies to identify interaction sites between the three assembly factors . Cryo-EM studies have revealed that ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, resulting in efflux of the FAD cofactor .

• Peptide competition assays: Synthetic peptides spanning ECSIT residues 318-336 have been shown to eject FAD from ACAD9, confirming these residues are crucial for complex formation . This approach can help map specific interaction domains.

• Dynamic Light Scattering (DLS) and mass photometry: These techniques have been used to analyze the behavior of ACAD9 mutants in regards to their ECSIT-binding properties .

What considerations are important when using ACAD9 antibodies to study disease-related mutations?

When studying ACAD9 mutations associated with complex I deficiency, several considerations are critical:

• Mutation mapping: Over 40 currently known pathogenic mutation sites have been mapped onto homology-modeled ACAD9 structures, providing structural insights into disease mechanisms . When designing experiments with FITC-conjugated ACAD9 antibodies, it's important to consider whether the epitope recognized by the antibody might be affected by specific mutations.

• Functional domains impact:

  • Mutations in the FAD binding site may affect both ACAD9's dehydrogenase activity and complex I assembly function

  • Mutations in the "gatekeeper loop" (residues near Gly186) may specifically impact ECSIT binding and FAD ejection, as this loop moves approximately 10 Å upwards during ECSIT binding

  • Mutations in the α-helix adjacent to the FAD-binding loop should be considered as key structural elements that specifically enable CI assembly functionality

• Experimental design considerations:

  • Use multiple antibodies targeting different epitopes to ensure detection regardless of mutation location

  • Complement protein detection with mRNA analysis, especially for mutations that might affect protein stability

  • Include functional assays alongside localization studies to correlate structural changes with functional outcomes

• ACAD9 activity measurement: For mutations like Arg469Trp, Arg518His, and Arg532Trp, which have shown similar dehydrogenation activities to wild-type ACAD9, complementary assays beyond antibody detection are necessary to understand the pathogenic mechanism .

What approaches can be used to study ACAD9 in specialized cellular models or tissues?

Different experimental models require tailored approaches when using FITC-conjugated ACAD9 antibodies:

• Mouse models:

  • Tissue-specific ACAD9 knockout mice have been developed that demonstrate symptoms based on the affected tissue

  • When using mouse models, confirm antibody cross-reactivity between human and mouse ACAD9

  • In conditional knockouts, consider using FITC-ACAD9 antibodies alongside Cre-recombinase markers to confirm deletion in specific tissues

• Fibroblast cultures:

  • Patient-derived fibroblasts have been extensively used in ACAD9 research

  • Cytokine stimulation of cultured human fibroblasts can modulate ACAD activity and should be considered when designing experiments

  • Flow cytometry with FITC-conjugated ACAD9 antibodies can quantify expression changes in response to treatments

• Liver and muscle preparations:

  • Protocols using 50 μg of total protein from human fibroblasts or liver extracts and 150 μg of total protein from muscle lysates have been established for immunoblotting

  • For immunofluorescence in tissue sections, consider tissue autofluorescence, particularly in liver tissue which may overlap with FITC emission

• Cell models with manipulated ACAD9 expression:

  • When studying promoter variants, complementary techniques like the luciferase reporter assays described in the literature should be employed

  • For transfection experiments, HepG2 cells have been successfully used to study ACAD9 expression regulation

How do I optimize ACAD9 immunofluorescence staining to reduce background?

Optimizing signal-to-noise ratio in FITC-conjugated ACAD9 antibody staining requires attention to several factors:

• Fixation optimization:

  • Excessive fixation can mask epitopes while insufficient fixation may compromise structural integrity

  • For mitochondrial proteins like ACAD9, a brief fixation (10-15 minutes) with 4% paraformaldehyde is often optimal

• Blocking strategies:

  • Use 5-10% normal serum from the same species as the secondary antibody when using indirect detection methods

  • For direct FITC-conjugated antibodies, employ species-matched normal serum or 1-3% BSA

  • Consider adding 0.1-0.3% Triton X-100 to blocking solutions for better penetration in fixed samples

• Autofluorescence reduction:

  • For tissues with high autofluorescence (liver, brain), consider treatments with sodium borohydride (0.1% for 5 minutes) or 0.1-1% Sudan Black B in 70% ethanol after antibody incubation

  • In cell culture models, shorter fixation times and careful washing can minimize autofluorescence

• Antibody optimization:

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

  • Increase incubation time at 4°C rather than increasing antibody concentration

  • Consider using amplification systems for weak signals rather than higher primary antibody concentrations

• Mitochondrial co-localization:

  • Use mitochondrial markers like MitoTracker (with a non-overlapping emission spectrum) to confirm specificity of ACAD9 staining

  • DAPI nuclear counterstain can help delineate cellular architecture while having minimal spectral overlap with FITC

What approaches can validate ACAD9 antibody specificity in experimental settings?

Validating ACAD9 antibody specificity is crucial for reliable experimental outcomes:

• Genetic validation approaches:

  • Use ACAD9 knockdown/knockout systems as negative controls

  • The tissue-specific ACAD9 knockout mouse models described in the literature provide excellent specificity controls

• Biochemical validation:

  • Perform immunocompetition assays by adding purified ACAD9 protein to the antibody incubation solution at a 1:1 molar ratio, as described in published protocols

  • Immunoinactivation assays using protein A-bound resin to precipitate antibody-antigen complexes can confirm specificity

• Cross-reactivity assessment:

  • Test against related proteins, particularly VLCAD which shares high sequence similarity with ACAD9

  • Compare ACAD9 antibody staining patterns with VLCAD and other ACAD family members

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of ACAD9 and compare staining patterns

  • Combine with mRNA detection methods like RT-PCR to correlate protein and transcript levels

• Functional correlation:

  • Correlate antibody staining intensity with functional assays of ACAD9 activity

  • Compare antibody detection to FAD binding capacity, as ACAD9 contains approximately 70% FAD compared to VLCAD

How can FITC-conjugated ACAD9 antibodies be used in multiplexed imaging applications?

Multiplexed imaging with FITC-conjugated ACAD9 antibodies requires careful experimental design:

• Compatible fluorophore selection:

  • FITC emission peaks at approximately 525 nm (green), allowing combination with fluorophores emitting in red (e.g., Cy3, Texas Red) and far-red (e.g., Cy5, Alexa Fluor 647) ranges

  • When studying ACAD9-ECSIT-NDUFAF1 interactions, consider using FITC for ACAD9, a red fluorophore for ECSIT, and a far-red fluorophore for NDUFAF1

• Sequential staining protocols:

  • For multiple primary antibodies from the same species, employ sequential staining with blocking steps

  • Consider using directly conjugated primary antibodies from different species to avoid cross-reactivity

• Advanced multiplexing techniques:

  • Spectral unmixing can resolve partially overlapping fluorescence emissions

  • For highly complex co-localization studies, consider employing cyclic immunofluorescence with FITC-conjugated ACAD9 antibodies as one of the detection rounds

• Multi-dimensional analysis:

  • Combine with z-stack confocal imaging to visualize spatial relationships of ACAD9 with binding partners

  • Time-lapse imaging with FITC-conjugated ACAD9 antibodies in permeabilized live cells can provide insights into dynamic interactions

• Quantification approaches:

  • Use colocalization coefficients (Pearson's, Mander's) to quantify spatial relationships

  • For flow cytometry applications, use appropriate compensation controls to account for spectral overlap

How should researchers quantify ACAD9 expression levels from immunofluorescence or flow cytometry data?

Accurate quantification of ACAD9 expression requires standardized approaches:

• Immunofluorescence quantification:

  • Use integrated density measurements normalized to cell area or mitochondrial markers

  • Employ standardized acquisition settings across all experimental conditions

  • Include calibration standards with known fluorophore concentrations for absolute quantification

• Flow cytometry quantification:

  • Report median fluorescence intensity (MFI) rather than mean values to minimize the impact of outliers

  • Use molecules of equivalent soluble fluorochrome (MESF) beads for standardization across experiments

  • Apply appropriate compensation when multiplexing with other fluorophores

• Western blot correlation:

  • Validate fluorescence intensity measurements with quantitative western blotting

  • Use the previously described protocols for immunoblotting with 50-150 μg of total protein depending on tissue type

• PCR validation:

  • Correlate protein expression with mRNA levels using quantitative PCR techniques

  • Similar to published research, normalize to housekeeping genes like GUSB or 18S rRNA using the ΔΔC_T method

• Standardization considerations:

  • Include lot-to-lot antibody validation to ensure consistent quantification

  • Maintain consistent cell culture conditions, as ACAD9 expression can be modulated by cytokine stimulation

What considerations are important when analyzing ACAD9 localization and co-localization data?

Accurate analysis of ACAD9 localization requires attention to several factors:

How can researchers interpret discrepancies between ACAD9 antibody results and functional data?

When faced with discrepancies between ACAD9 detection and functional outcomes, consider these analytical approaches:

• Dual functionality assessment:

  • Remember that ACAD9 has dual roles in FAO and complex I assembly, which may be differentially affected

  • The interaction with ECSIT causes FAD ejection from ACAD9, resulting in loss of FAO activity while maintaining assembly function

• Post-translational modifications:

  • Consider whether the antibody epitope might be affected by post-translational modifications

  • Analyze whether functional changes might result from modifications rather than expression changes

• Protein complexes:

  • ACAD9 exists in different protein complexes that may mask epitopes

  • The binary complex of ACAD9 with ECSIT is unstable and aggregates easily, while the ternary complex with NDUFAF1 is stable

• Conformational changes:

  • ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9

  • These structural changes may affect antibody recognition depending on epitope location

• Analytical approach:

  • Use multiple antibodies targeting different epitopes

  • Combine with mass spectrometry-based approaches for unbiased protein quantification

  • Consider native versus denatured detection methods to account for conformational states

What are the optimal experimental designs for studying ACAD9 interactions with ECSIT and NDUFAF1?

For investigating ACAD9-ECSIT-NDUFAF1 interactions, consider these experimental approaches:

• Co-immunoprecipitation with FITC detection:

  • Use FITC-conjugated ACAD9 antibodies for direct visualization of pull-down complexes

  • Remember that while the binary ACAD9-ECSIT complex is unstable, the ternary complex with NDUFAF1 is stable and soluble

• Binding domain mapping:

  • ECSIT binds to the carboxy-terminal half of ACAD9

  • NDUFAF1 binds to the amino-terminal half of ECSIT

  • Design experiments to visualize these interactions using domain-specific antibodies or tagged constructs

• Structural analysis integration:

  • Incorporate findings from structural studies showing that ECSIT binding induces a conformational change in the FAD-binding loop of ACAD9

  • Use peptide competition assays with synthetic peptides spanning ECSIT residues 318-336, which have been shown to eject FAD from ACAD9

• Dynamic interaction studies:

  • Design FRET-based approaches using FITC-conjugated ACAD9 antibodies paired with compatible acceptor fluorophores on ECSIT or NDUFAF1 antibodies

  • Consider time-resolved studies to capture assembly kinetics

• Mutational analysis:

  • Include experiments with ACAD9 mutants that affect the "gatekeeper loop" which undergoes a ~10 Å conformational change during ECSIT binding

  • Compare with VLCAD, which does not bind ECSIT, to identify critical structural differences

How should researchers design experiments to distinguish ACAD9 from other ACAD family members?

To ensure specificity when studying ACAD9 rather than related ACAD family proteins:

• Antibody selection strategies:

  • Choose antibodies targeting regions with lowest homology to VLCAD and other ACAD family members

  • Validate specificity using knockout models or recombinant protein competition

• Functional discrimination:

  • Only ACAD9 binds ECSIT and participates in complex I assembly

  • ACAD9 shows weaker FAD binding (70% occupancy) and lower dehydrogenase activity (18% of VLCAD activity)

  • Design assays leveraging these functional differences

• Structural differences to target:

  • Focus on the α-helix adjacent to the FAD-binding loop, identified as a key structural element specifically enabling the CI assembly functionality of ACAD9

  • Consider the FAD binding pocket, which is larger in ACAD9 than VLCAD, leading to fewer and weaker bonding interactions

• Expression pattern analysis:

  • Compare tissue distribution and expression levels

  • Include multiple family members in parallel analyses to demonstrate specificity

• Binding partner verification:

  • Use ECSIT and NDUFAF1 co-localization as a specific identifier for ACAD9

  • Include VLCAD and other ACAD family members as negative controls

What experimental controls are essential when using ACAD9 antibodies in disease models?

When applying FITC-conjugated ACAD9 antibodies to disease models, incorporate these essential controls:

• Genetic background controls:

  • Include isogenic wild-type controls matched to disease models

  • For mouse models, use same-strain wild-type mice alongside tissue-specific knockout models

• Expression level validation:

  • Correlate antibody staining with mRNA quantification

  • Western blot analysis using established protocols (50-150 μg protein based on tissue type)

• Pathway validation:

  • Assess both FAO pathway function and complex I assembly

  • Remember that ACAD9-ECSIT interaction leads to FAD ejection and loss of FAO activity

• Mutant-specific controls:

  • For studies of pathogenic mutations, include both wild-type and known pathogenic controls

  • Consider the location of over 40 mapped pathogenic mutations in relation to antibody epitopes

• Tissue-specific considerations:

  • Account for differences in mitochondrial content across tissues

  • In tissue-specific knockouts, confirm deletion using genetic markers alongside antibody staining

• Technical controls:

  • Include absorption controls with recombinant ACAD9 protein

  • For immunoinactivation assays, follow established protocols using 1 μg purified antibody per 10 μg of liver lysate protein