OPA1 Antibody, FITC conjugated

What is OPA1 protein and why is it important in research?

OPA1 is a nuclear-encoded mitochondrial protein with similarity to dynamin-related GTPases that localizes to the inner mitochondrial membrane. It plays critical roles in regulating mitochondrial stability, energy output, and sequestering cytochrome c. OPA1 protects cells from apoptosis by regulating inner membrane dynamics . The importance of OPA1 in research stems from its association with dominant optic atrophy, a degeneration of retinal ganglion cells, and its role in neurodegenerative conditions like Parkinson's disease where it serves as a molecular link between complex I deficiency and alterations in mitochondrial dynamics . The FITC-conjugated antibody enables direct fluorescent detection of OPA1 in various experimental settings.

What are the key applications of FITC-conjugated OPA1 antibodies?

FITC-conjugated OPA1 antibodies are valuable tools in multiple applications:

The FITC conjugation (excitation = 495 nm, emission = 519 nm) eliminates the need for secondary antibody incubation, reducing background signals and experimental time .

What are the storage and handling recommendations for FITC-conjugated OPA1 antibodies?

For optimal performance and extended shelf-life of FITC-conjugated OPA1 antibodies:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles that can degrade both antibody activity and fluorophore brightness

• Store at 4°C in the dark after thawing to prevent photobleaching of the FITC fluorophore

• Aliquot antibodies when possible to minimize freeze-thaw cycles

• Use proper buffer conditions (typically PBS with 0.03% Proclin 300 and 50% Glycerol, pH 7.4) to maintain stability

• Protect from prolonged exposure to light during experiments to prevent fluorophore bleaching

What dilutions are recommended for different applications of FITC-conjugated OPA1 antibodies?

The optimal antibody dilution varies by application and manufacturer. Below are general recommendations based on the search results:

Always perform a dilution series to determine the optimal concentration for your specific experimental conditions, cell types, and tissue samples. Signal-to-noise ratio should be evaluated to determine the most appropriate dilution.

What tissue and cell types have been validated for OPA1 antibody reactivity?

OPA1 antibodies have demonstrated reactivity across multiple species and sample types:

The antibody has been validated for detection of OPA1 in various cellular compartments, particularly in mitochondrial inner membrane and intermembrane space .

How can FITC-conjugated OPA1 antibodies be used to investigate mitochondrial dynamics in neurodegenerative diseases?

FITC-conjugated OPA1 antibodies provide valuable tools for studying mitochondrial dynamics in neurodegenerative diseases like Parkinson's disease (PD). Research has shown that complex I inhibition by parkinsonian neurotoxins leads to oxidative-dependent disruption of OPA1 oligomeric complexes that normally maintain tight mitochondrial cristae junctions .

Methodological approach:

• Use FITC-conjugated OPA1 antibodies for live-cell imaging to monitor OPA1 distribution before and after treatment with mitochondrial toxins

• Combine with mitochondrial markers to assess colocalization and structural changes

• Implement high-resolution microscopy techniques (super-resolution, confocal) to visualize cristae remodeling

• Quantify changes in OPA1 oligomerization state using biochemical approaches alongside immunofluorescence

• Track mitochondrial morphology changes temporally using time-lapse microscopy in toxin-treated versus control cells

Researchers have demonstrated that OPA1 overexpression can abrogate mitochondrial structural remodeling and dopaminergic neurodegeneration both in vitro and in vivo, indicating OPA1 as a potential therapeutic target for complex I cytopathies such as PD .

What are the key considerations for dual immunofluorescence studies involving FITC-conjugated OPA1 antibodies?

When designing dual or multi-color immunofluorescence studies:

• Fluorophore selection: Choose secondary fluorophores with minimal spectral overlap with FITC (excitation = 495 nm, emission = 519 nm) . Compatible partners include:

  • Cy3 (excitation ~550 nm, emission ~570 nm)

  • Alexa Fluor 594/647 (minimal spectral overlap)

  • DAPI for nuclear counterstaining

• Sample processing:

  • For fixed cells: Use 2-4% paraformaldehyde fixation followed by permeabilization with 0.1-0.5% Triton X-100

  • For tissues: Optimize antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Signal optimization:

  • Apply sequential scanning in confocal microscopy to minimize bleed-through

  • Include single-stained controls for spectral unmixing

  • Adjust antibody concentrations to achieve comparable signal intensities across channels

• Controls:

  • Include a negative control by omitting primary antibody

  • Validate specificity using OPA1 knockdown/knockout samples

  • Include mitochondrial markers (MitoTracker, Tom20) for colocalization validation

How can researchers distinguish between different OPA1 isoforms using FITC-conjugated antibodies?

OPA1 undergoes complex posttranscriptional regulation and posttranslational proteolysis, resulting in multiple isoforms. The gene product can be cleaved into chains with molecular weights of approximately 100 kDa and 80-90 kDa .

Methodological approach:

• Western blot analysis: Use gel electrophoresis conditions that maximize separation in the 80-100 kDa range

• Isoform characterization:

  • Long forms (L-OPA1): ~100 kDa

  • Short forms (S-OPA1): 80-90 kDa

• Experimental controls:

  • Include samples with known OPA1 processing states (e.g., CCCP-treated cells show increased short forms)

  • Use isoform-specific inhibitors of OPA1 processing proteases (e.g., OMA1, YME1L inhibitors)

• Quantitative analysis:

  • Measure the L-OPA1:S-OPA1 ratio to assess mitochondrial stress conditions

  • Compare patterns across different cell types or disease models

For challenging distinctions, combining immunofluorescence with super-resolution microscopy can help visualize the subcellular distribution of different OPA1 forms, though FITC-conjugated antibodies may not distinguish between specific isoforms without additional techniques.

What are common technical challenges when using FITC-conjugated OPA1 antibodies and how can they be addressed?

How should researchers interpret changes in OPA1 localization and expression patterns in disease models?

When analyzing OPA1 localization and expression patterns:

• Normal patterns:

  • OPA1 typically shows punctate or reticular staining consistent with mitochondrial networks

  • Both long and short forms are present in healthy cells, with predominance of long forms

  • OPA1 maintains cristae structure through oligomeric complexes

• Disease-associated changes:

  • In Parkinson's disease models: Complex I inhibition leads to disruption of OPA1 oligomeric complexes, causing cristae disintegration, loss of matrix density, and mitochondrial swelling

  • These structural changes correlate with mobilization of cytochrome c from cristae to intermembrane space, lowering the threshold for apoptosis activation

• Quantitative assessment:

  • Measure mitochondrial morphology parameters (length, branching, circularity)

  • Quantify colocalization with other mitochondrial markers

  • Analyze the ratio of fusion/fission events in live cell imaging

• Functional correlations:

  • Associate changes in OPA1 distribution with measurements of mitochondrial membrane potential

  • Correlate with respiratory capacity and ATP production

  • Link structural changes to cell death markers and cytochrome c release

Research has demonstrated that OPA1 overexpression can reverse mitochondrial abnormalities and prevent neurodegeneration in PD models, suggesting therapeutic potential .

What controls should be included when validating the specificity of FITC-conjugated OPA1 antibodies in new experimental systems?

Comprehensive validation of FITC-conjugated OPA1 antibodies requires multiple controls:

• Genetic controls:

  • OPA1 knockdown/knockout samples: Should show significant reduction in signal

  • OPA1 overexpression: Should show increased signal intensity

  • Use siRNA/shRNA with different targeting sequences to confirm specificity

• Biological controls:

  • Include multiple cell types with known OPA1 expression (e.g., HeLa, HEK-293, neuronal cells)

  • Compare mitochondria-rich versus mitochondria-poor cell types

  • Test across species if cross-reactivity is claimed (human, mouse, rat, etc.)

• Technical controls:

  • Peptide competition assay: Pre-incubation with immunizing peptide should abolish specific signal

  • Omit primary antibody: Assess background from secondary reagents or autofluorescence

  • Colocalization with independent mitochondrial markers (e.g., MitoTracker, Tom20)

  • Compare with alternative OPA1 antibody clones/sources

• Application-specific controls:

  • For Western blot: Confirm molecular weight (80-100 kDa)

  • For IHC: Include tissue types with varying OPA1 expression levels

  • For live-cell imaging: Compare with fixed-cell results to confirm patterns

How can FITC-conjugated OPA1 antibodies be used to study mitochondrial cristae remodeling during apoptosis?

OPA1 plays a critical role in maintaining cristae junctions and sequestering cytochrome c. During apoptosis, OPA1 oligomeric complexes are disrupted, leading to cristae remodeling and cytochrome c release .

Methodological approach:

• Experimental design:

  • Induce apoptosis using established triggers (staurosporine, TNFα, complex I inhibitors)

  • Use time-course experiments to capture progressive cristae remodeling

  • Combine with cytochrome c staining to correlate structural changes with release events

• Imaging techniques:

  • Super-resolution microscopy (STED, PALM, STORM) to visualize cristae structure

  • Correlative light and electron microscopy (CLEM) to link fluorescence patterns with ultrastructural changes

  • Live-cell imaging with gentle acquisition parameters to minimize phototoxicity

• Quantitative analysis:

  • Measure OPA1 oligomerization state through biochemical methods alongside imaging

  • Quantify cristae width and junction diameter from electron micrographs

  • Analyze the temporal relationship between OPA1 disruption and cytochrome c release

• Interventional approaches:

  • Express non-cleavable OPA1 mutants to prevent cristae remodeling

  • Use antioxidants to determine the role of oxidative stress in OPA1 complex disruption

  • Manipulate proteases involved in OPA1 processing (OMA1, YME1L)

Research has shown that OPA1 overexpression can protect against mitochondrial structural remodeling and neurodegeneration in PD models, highlighting the therapeutic potential of targeting this pathway .

What are the considerations for using FITC-conjugated OPA1 antibodies in live-cell imaging experiments?

Live-cell imaging with FITC-conjugated antibodies presents specific challenges:

• Cell permeabilization strategies:

  • Gentle permeabilization with digitonin (10-25 μg/ml) to selectively permeabilize plasma membrane

  • Protein delivery systems (Chariot, BioPORTER) to introduce antibodies without fixation

  • Cell-penetrating peptide conjugates for enhanced antibody internalization

• Phototoxicity and bleaching mitigation:

  • Minimize exposure time and intensity (use ND filters)

  • Employ oxygen scavengers in imaging media (Oxyrase, PCA/PCD system)

  • Use pulse-illumination strategies with longer recovery periods

  • Consider alternative platforms like spinning disk confocal to reduce light exposure

• Controls and validation:

  • Confirm mitochondrial targeting with established live mitochondrial dyes (MitoTracker)

  • Monitor cell health parameters throughout imaging session

  • Compare patterns with fixed-cell immunofluorescence to validate specificity

• Technical limitations:

  • FITC conjugates may not be optimal for extended live imaging due to photobleaching

  • Consider more photostable alternatives (Alexa Fluor 488) for lengthy experiments

  • Be aware that antibody binding may interfere with protein function in live cells