OCIAD1 Antibody

What is OCIAD1 and what cellular functions does it serve?

OCIAD1 is an inner mitochondrial membrane protein that forms a complex with supramolecular prohibitin assemblies. Research has identified OCIAD1 as a Complex III assembly factor in the mitochondrial electron transport chain (mETC) . It functions as an adaptor within prohibitin assemblies to stabilize and/or chaperone cytochrome c1 (CYC1) and facilitate its proteolytic processing by the IMMP2L protease . OCIAD1 is crucial for maintaining normal steady-state levels of Complex III, and its knockdown results in compromised Complex III assembly and sensitizes cells specifically to Complex III inhibition . The protein has also been implicated in cancer biology, with aberrant expression observed in ovarian carcinomas .

What should researchers consider when selecting an OCIAD1 antibody?

When selecting an OCIAD1 antibody, researchers should consider:

• Target species reactivity: Ensure the antibody is validated for your species of interest. For example, some antibodies like the OCIAD1 Polyclonal Antibody (CAB11630) are specifically reactive with human samples .

• Application compatibility: Verify the antibody has been validated for your intended application (Western blot, immunofluorescence, immunoprecipitation, etc.) .

• Clonality: Polyclonal antibodies like CAB11630 offer high sensitivity and recognize multiple epitopes, making them useful for detecting low-abundance proteins, while monoclonal antibodies provide higher specificity for a single epitope .

• Validation data: Review available validation data, including Western blot images showing appropriate band patterns and specificity testing in relevant experimental systems.

• Epitope location: Consider whether the antibody's epitope is located in a region that might be masked in protein complexes or affected by post-translational modifications.

How should OCIAD1 antibodies be validated before use in critical experiments?

Thorough validation is essential before using OCIAD1 antibodies in critical experiments:

• Positive and negative controls: Use cells/tissues known to express or lack OCIAD1. Knockdown or knockout models serve as excellent negative controls.

• Multiple detection methods: Confirm findings using different techniques (Western blot, immunofluorescence, etc.).

• Protein band verification: For Western blot applications, verify that the detected band corresponds to the expected molecular weight of OCIAD1 (~27-30 kDa).

• Cross-reactivity testing: Test for potential cross-reactivity with similar proteins, particularly OCIAD2, which shares sequence similarity but has different functions .

• Lot-to-lot consistency: If changing antibody lots, perform parallel validation to ensure consistent results.

What is the optimal protocol for detecting OCIAD1 in Western blot applications?

Based on research practices with OCIAD1:

Sample Preparation:

• For whole-cell lysates: Lyse cells in RIPA buffer supplemented with protease inhibitors.

• For mitochondrial enrichment: Use selective digitonin permeabilization or differential centrifugation methods .

SDS-PAGE and Transfer:

• Load 20-40 μg protein per lane on 10-12% polyacrylamide gels.

• Transfer to PVDF membrane (preferred over nitrocellulose for mitochondrial proteins).

Immunoblotting:

• Block with 5% non-fat milk in TBST for 1 hour at room temperature.

• Incubate with primary OCIAD1 antibody (e.g., CAB11630) at 1:1000 dilution overnight at 4°C .

• Wash 3× with TBST, 10 minutes each.

• Incubate with appropriate HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature.

• Wash 3× with TBST, 10 minutes each.

• Develop using ECL substrate and image.

Expected Results:

• Primary band at ~27-30 kDa (mature OCIAD1)

• Possible additional bands representing precursor forms or post-translationally modified variants

How can I use OCIAD1 antibodies to study its interaction with prohibitin complexes?

OCIAD1 forms complexes with prohibitin assemblies in the inner mitochondrial membrane . To study these interactions:

Co-immunoprecipitation (Co-IP):

• Prepare mitochondrial lysates using mild detergents like LMNG (lauryl maltose neopentyl glycol) to preserve protein-protein interactions .

• Perform immunoprecipitation using anti-OCIAD1 antibody coupled to protein A/G beads.

• Wash the immunoprecipitates thoroughly.

• Analyze by Western blot for the presence of prohibitin complex components (PHB1, PHB2).

Blue Native PAGE (BN-PAGE):

• Solubilize mitochondria with LMNG to maintain native protein complexes .

• Separate on 3-12% gradient native PAGE gels.

• Transfer to PVDF membrane.

• Probe with anti-OCIAD1 and anti-prohibitin antibodies.

• Look for co-migration at approximately 0.9-1 MDa, which indicates OCIAD1-prohibitin complexes .

In-gel Mobility Shift Assay:

• Pre-incubate LMNG-solubilized mitochondrial membranes with anti-PHB2 antibody or vehicle control.

• Run on BN-PAGE.

• Perform Western analysis with anti-PHB2 and anti-OCIAD1 antibodies.

• A retarded migration of both PHB2 and OCIAD1 assemblies indicates association .

What is the best approach to visualize OCIAD1 subcellular localization?

To visualize OCIAD1's mitochondrial localization:

Immunofluorescence Protocol:

• Fix cells with 4% paraformaldehyde for 15 minutes.

• Permeabilize with 0.2% Triton X-100 for 10 minutes.

• Block with 3% BSA for 1 hour.

• Co-stain with:

  • Anti-OCIAD1 antibody (1:200 dilution)

  • Mitochondrial marker (e.g., MitoTracker or anti-TOMM20)

  • Nuclear stain (e.g., DAPI)

• Apply appropriate secondary antibodies.

• Image using confocal microscopy.

Split-GFP Complementation:
An alternative approach used in research involves split-GFP complementation assays:

• Clone OCIAD1 into a pGFP11-N1 or pGFP11-C1 vector .

• Co-express with mitochondrial marker proteins tagged with complementary GFP fragments.

• GFP fluorescence will be reconstituted only where OCIAD1 interacts with or is proximal to the marker protein.

How can I design experiments to study OCIAD1's role in Complex III assembly?

To investigate OCIAD1's role in Complex III assembly:

BN-PAGE Analysis:

• Isolate mitochondria from control and OCIAD1 knockdown cells (using CRISPRi or siRNA) .

• Separate protein complexes using BN-PAGE.

• Perform Western blot analysis using antibodies against core subunits of respiratory Complexes I-V.

• Focus on comparing the levels of:

  • Complex III dimer (CIII₂)

  • Supercomplexes containing Complex III (CIII₂CIV, CICIII₂, CICIII₂CIV)

Rescue Experiments:

• In OCIAD1 knockdown cells, reintroduce:

  • Wild-type OCIAD1

  • OCIAD1 mutants (e.g., F102A point mutant or Δ97-115 truncation)

• Assess Complex III assembly by BN-PAGE as above.

• Compare with control cells to evaluate rescue efficiency.

Respiratory Chain Function Assays:

• Measure oxygen consumption rate (OCR) using a Seahorse XF analyzer.

• Assess specific Complex III activity using spectrophotometric assays.

• Compare control, OCIAD1 knockdown, and rescue cell lines.

How can I investigate OCIAD1's role in CYC1 processing?

OCIAD1 is required for normal processing of cytochrome c1 (CYC1) . To study this role:

Western Blot Analysis of CYC1 Processing:

• Prepare digitonin-solubilized mitochondria from control and OCIAD1 knockdown cells.

• Separate proteins using SDS-PAGE.

• Probe with anti-CYC1 antibody to detect:

  • Mature CYC1 (~30 kDa)

  • Intermediate form (after MPP processing)

  • Precursor form

CYC1 Hemylation Assessment:

• Run mitochondrial fractions on SDS-PAGE.

• Assess hemylation using chemiluminescence detection method to visualize heme-containing proteins .

• Compare the hemylation status of all CYC1 forms across samples.

Mass Spectrometry Analysis:

• Excise BN-PAGE gel slices containing CIII₂ assemblies.

• Perform tryptic digestion and LC-MS/MS analysis.

• Look for specific peptides representing:

  • Mature CYC1 N-terminus (e.g., SDLELHPPSYPWSHR)

  • CYC1 precursor fragments (e.g., TPQAVALSSK)

• Compare peptide abundance between control and OCIAD1 knockdown samples.

What methods can I use to create and validate OCIAD1 genetic models?

To develop genetic models for studying OCIAD1 function:

CRISPR/Cas9 Knockout or Knockdown:

• Design sgRNAs targeting OCIAD1 (validated sgRNAs include sgRNA#2 used in published research) .

• For stable knockdown, use CRISPRi systems with dCas9-KRAB.

• Validate knockdown efficiency by Western blot using anti-OCIAD1 antibodies.

• Assess phenotypes including:

  • Growth defects in galactose media

  • Sensitivity to Complex III inhibitors (e.g., antimycin A)

  • Complex III assembly using BN-PAGE

Expression of OCIAD1 Variants:

• Generate lentiviral vectors expressing:

  • Wild-type OCIAD1

  • OCIAD1 point mutants (e.g., F102A)

  • OCIAD1 truncations (e.g., Δ97-115)

• Include appropriate tags (GFP, StrepII) for detection and purification.

• Express in OCIAD1 knockdown backgrounds to assess rescue capability.

Verification Methods:

• Western blot with anti-OCIAD1 antibodies

• qRT-PCR for transcript levels

• Functional assays for mitochondrial respiratory activity

• BN-PAGE for Complex III assembly status

Why might I detect multiple bands when probing for OCIAD1 in Western blots?

Multiple bands when detecting OCIAD1 could indicate:

• Different processing forms: OCIAD1 might exist in precursor, intermediate, and mature forms, similar to what is observed with CYC1 .

• Post-translational modifications: Modifications like phosphorylation or ubiquitination could cause mobility shifts.

• Alternative splicing variants: Different isoforms of OCIAD1 may be expressed in certain cell types.

• Proteolytic degradation: Partial degradation during sample preparation may generate fragments.

• Non-specific binding: The antibody might cross-react with similar proteins like OCIAD2 or other related proteins .

Verification approaches:

• Compare band patterns in control vs. OCIAD1 knockdown samples

• Use different antibodies targeting different epitopes

• Perform peptide competition assays

• Include positive controls with tagged OCIAD1 constructs of known sizes

How can I address inconsistent OCIAD1 detection in my experiments?

If you're experiencing inconsistent OCIAD1 detection:

Sample Preparation Considerations:

• Ensure complete protease inhibition during sample preparation.

• Optimize mitochondrial isolation protocols - OCIAD1 is predominantly localized to mitochondria .

• Test different lysis buffers (RIPA vs. milder detergents like LMNG for native conditions) .

• Avoid freeze-thaw cycles of prepared samples.

Antibody-Related Factors:

• Titrate antibody concentration to determine optimal working dilution.

• Test different antibody lots or sources.

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

• Consider using fresh antibody aliquots to avoid degradation.

Detection System Optimization:

• For low-abundance detection, use high-sensitivity ECL substrates.

• Optimize exposure times to avoid saturation or underexposure.

• Consider fluorescent secondary antibodies for more quantitative results.

Experimental Design Controls:

• Always include positive and negative controls.

• Use OCIAD1 knockout/knockdown samples as specificity controls .

• Include recombinant OCIAD1 as a reference standard.

How should I interpret contradictory findings about OCIAD1 function in my research?

When facing contradictory data about OCIAD1 function:

Systematic Approach to Reconciling Contradictions:

• Examine experimental conditions:

  • Cell type differences (cancer vs. normal cells)

  • Growth media composition (glucose vs. galactose)

  • Level of OCIAD1 depletion (partial vs. complete)

• Consider compensatory mechanisms:

  • OCIAD2 upregulation in OCIAD1-deficient cells

  • Alternative pathways for Complex III assembly

  • Adaptations during long-term vs. acute OCIAD1 depletion

• Review technical variables:

  • Methods used for assessing mitochondrial function

  • Assay sensitivity and dynamic range

  • Timepoints examined (immediate vs. long-term effects)

• Design experiments to directly test contradictions:

  • Side-by-side comparison of different cell lines

  • Time-course experiments

  • Use of multiple independent methods to assess the same parameter

Data integration framework:

What advanced techniques can help resolve complex questions about OCIAD1 function?

For resolving complex questions about OCIAD1:

Proximity Labeling Techniques:

• BioID or TurboID fusion with OCIAD1 to identify proximal proteins in living cells

• APEX2-OCIAD1 fusion for electron microscopy visualization of precise submitochondrial localization

Structural Biology Approaches:

• Cryo-EM analysis of prohibitin-OCIAD1 complexes

• Cross-linking mass spectrometry to map specific interaction sites between OCIAD1 and binding partners

Live-Cell Imaging:

• FRAP (Fluorescence Recovery After Photobleaching) of fluorescently tagged OCIAD1 to assess dynamics

• Split-GFP complementation assays to visualize OCIAD1 interactions with specific partners in real-time

Genetic Interaction Mapping:

• CRISPR interference screens in the presence of antimycin A to identify synthetic lethal or suppressor interactions with OCIAD1

• Double knockdown experiments testing OCIAD1 with other Complex III assembly factors

Metabolic Profiling:

• Targeted metabolomics to measure levels of TCA cycle intermediates

• Isotope tracing to assess metabolic flux through mitochondrial pathways in OCIAD1-deficient cells

What are the emerging roles of OCIAD1 beyond mitochondrial function?

While OCIAD1's role in mitochondrial Complex III assembly is established , researchers should be aware of potential additional functions:

• Cancer biology: OCIAD1 has been implicated in ovarian carcinomas, suggesting roles in cancer progression or metabolism .

• Potential signaling functions: The protein may participate in cellular signaling pathways beyond its structural role in mitochondria.

• Interaction with other cellular compartments: OCIAD1 might have functions beyond the mitochondria, possibly through splicing variants or moonlighting activities.

• Stress response: Given its role in mitochondrial function, OCIAD1 may be involved in cellular stress responses and adaptation.

What considerations should guide future OCIAD1 antibody development?

For researchers developing or selecting next-generation OCIAD1 antibodies:

• Epitope selection for specific applications:

  • Antibodies targeting the OCIA domain (residues 97-115) for studying functions dependent on this region

  • Antibodies against N-terminal regions to detect all forms of OCIAD1

  • Non-overlapping epitope antibodies for sandwich assays

• Application-optimized antibodies:

  • Native condition-compatible antibodies for IP applications

  • Fixed-tissue compatible antibodies for IHC/IF

  • Antibodies that can distinguish between OCIAD1 and the related OCIAD2

• Tag-specific considerations:

  • Antibodies compatible with various fusion constructs (GFP-OCIAD1, OCIAD1-StrepII, etc.)

  • Non-interfering antibodies that don't disrupt OCIAD1's interactions with prohibitins or Complex III components

• Validation standards:

  • Validation in multiple cell types

  • Demonstration of specificity using OCIAD1 knockout models

  • Cross-validation with orthogonal detection methods

By addressing these comprehensive considerations, researchers can maximize the utility and reliability of OCIAD1 antibodies in their investigations of this important mitochondrial protein.