OPA1 Antibody

What is OPA1 and what is its biological significance?

OPA1 exists in multiple isoforms - humans have eight different isoforms while mice have five. These isoforms are expressed as either short-form or long-form variants and contribute to OPA1's ability to control mitochondrial energetics and DNA maintenance . Defects in OPA1 are associated with optic atrophy type 1, leading to vision loss. The protein is predominantly expressed in the retina, although also found in the brain, testis, heart, and skeletal muscles .

What types of OPA1 antibodies are commercially available for research?

Several types of OPA1 antibodies are available for research applications:

These antibodies offer versatility for different experimental applications, with options for various detection systems .

For which applications are OPA1 antibodies validated?

OPA1 antibodies have been validated for multiple experimental applications:

• Western Blotting (WB): Both monoclonal and polyclonal OPA1 antibodies can detect OPA1 protein in cell or tissue lysates, typically appearing as bands of approximately 80-90 kDa .

• Immunoprecipitation (IP): OPA1 antibodies can isolate OPA1 protein from complex mixtures .

• Immunofluorescence (IF): These antibodies effectively visualize the subcellular localization of OPA1 protein in fixed cells or tissue sections .

• Enzyme-linked Immunosorbent Assay (ELISA): OPA1 antibodies have been validated for quantitative detection of OPA1 protein in samples .

• Immunohistochemistry (IHC): OPA1 antibodies can visualize OPA1 distribution in tissue sections, particularly useful for studying retinal ganglion cells and optic nerve .

The selection of the appropriate application depends on your research question and the specific properties of the antibody.

How should I optimize western blot conditions to visualize multiple OPA1 isoforms?

Optimizing western blot conditions is crucial for detecting multiple OPA1 isoforms. Based on recent research, the following protocol is recommended:

• Sample preparation:

  • Lyse cells or tissues in appropriate lysis buffer with protease inhibitors

  • Centrifuge at 7245 RCF for 15 minutes

  • Load 20-50 μg of lysate with 2X Laemmli sample buffer containing BME

• Gel electrophoresis:

  • Use gradient gels (4%-20% Tris-glycine gels) pre-chilled for 1 hour

  • Run at 100V for 10 minutes to move samples through wells

  • Reduce voltage to 50V and run for 5-7 hours at 4°C

• Transfer and detection:

  • Standard transfer to membrane

  • Use appropriate primary antibody (e.g., BD Biosciences OPA1 antibody)

  • Extend primary antibody incubation time if bands are weak

This optimized protocol has been shown to successfully detect up to five different OPA1 isoforms, with the top two bands representing long forms and the bottom three bands representing short isoforms .

What controls should I include when conducting immunohistochemistry with OPA1 antibodies?

When performing immunohistochemistry with OPA1 antibodies, the following controls are essential:

• Specificity controls:

  • Pre-absorb OPA1 antibody with specific blocking peptide (5 μg/ml corresponding to amino acids 938-960 of mOPA1)

  • Alternatively, pre-absorb with recombinant OPA1 protein (5 μg/ml)

  • Compare with non-pre-absorbed antibody; specific signal should be reduced in pre-absorbed samples

• Positive tissue controls:

  • Include tissues known to express OPA1 (e.g., retina, brain)

  • Verify the expected pattern of expression in these tissues

• Co-localization controls:

  • Perform double immunohistochemistry with markers such as:

    • Anti-neurofilament 200 (for neurons)

    • Anti-cytochrome c (for mitochondria)

    • Anti-GFAP (for astrocytes)

    • O4 antibody (for oligodendrocytes)

These controls help ensure that observed signals are specific to OPA1 rather than artifacts or non-specific binding.

What are the best fixation methods for preserving OPA1 detection in immunocytochemistry?

For optimal OPA1 detection in immunocytochemistry, the following fixation protocols have been empirically determined:

• For mitochondrial morphology preservation:

  • Fix cells with 0.5% glutaraldehyde for 30 minutes at 4°C

  • Quench autofluorescence with 1% sodium borohydride (30 minutes at room temperature)

  • Permeabilize with 0.1% Triton X-100/PBS (15 minutes at room temperature)

• For signal amplification:

  • After fixation and permeabilization, use Tyramide Signal Amplification Kit according to manufacturer's instructions

  • This approach enhances detection sensitivity while maintaining specificity

• For antibody incubation:

  • Use OPA1 antibody at appropriate dilution (e.g., 1:1,000 for polyclonal antibodies)

  • Incubate for 16 hours at 4°C for optimal binding

  • For secondary antibodies, goat anti-mouse IgG conjugated with Alexa Fluor 594 or similar fluorophores (1:200) work well

These fixation and detection methods have been validated for retinal ganglion cells and RGC-5 cells but may require optimization for other cell types.

Why am I unable to detect OPA1 bands in my western blot?

If you're experiencing difficulties detecting OPA1 bands in western blots, consider these common issues and solutions:

Remember that OPA1 typically appears as 2-5 bands between 80-90 kDa, with the pattern potentially varying between tissues and species .

How can I verify the specificity of my OPA1 antibody?

Verifying antibody specificity is crucial for reliable OPA1 research. Implement these verification strategies:

• Immunoblot verification:

  • Run recombinant OPA1 protein alongside experimental samples

  • Verify the characteristic banding pattern (approximately 80 and 90 kDa bands)

  • Compare band patterns across different tissue types

• Pre-absorption controls:

  • Incubate OPA1 antibody with specific blocking peptide (5 μg/ml)

  • Alternatively, pre-absorb with recombinant OPA1 protein (5 μg/ml)

  • Specific binding should be significantly reduced in pre-absorbed samples

• Cross-reactivity assessment:

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

  • Compare expression patterns with published literature

Implementing these verification steps ensures that observed signals are due to specific detection of OPA1 rather than non-specific binding.

What challenges exist in differentiating long and short OPA1 isoforms?

Differentiating between long and short OPA1 isoforms presents several technical challenges:

• Close molecular weights:

  • OPA1 isoforms have relatively similar sizes (approximately 80-90 kDa)

  • Standard electrophoresis conditions often fail to separate these isoforms

  • Solution: Use gradient gels with extended run times at lower voltage

• Isoform complexity:

  • Humans express eight different OPA1 isoforms, mice express five

  • These result from both alternative splicing and proteolytic processing

  • The top two bands typically represent long forms, while bottom three bands are short isoforms

• Tissue-specific expression:

  • The abundance of specific isoforms varies across tissues and cell types

  • Analysis may require optimization for each experimental model

  • Some antibodies may have differential sensitivity to specific isoforms

Advanced researchers have successfully visualized up to six bands by optimizing gradient gel conditions, particularly for detecting cleavage of S3 isoforms at the S3 domain .

What is the expression pattern of OPA1 in neural tissues?

OPA1 shows distinct expression patterns in neural tissues, particularly in the visual system:

• Retina:

  • OPA1 is predominantly expressed in retinal ganglion cells

  • It localizes to mitochondria in both cell bodies and processes

  • This expression pattern helps explain the selective vulnerability of retinal ganglion cells to OPA1 mutations

• Optic nerve:

  • OPA1 protein is present in axonal mitochondria throughout the optic nerve

  • Expression is detected in both immunoblot and immunohistochemical analyses

  • OPA1 is critical for maintaining mitochondrial function in these high-energy demanding axons

• Brain:

  • Western blot analysis shows OPA1 expression in brain tissue

  • Similar to retina and optic nerve, brain tissue shows two major OPA1 bands (approximately 90 and 80 kDa)

• Expression in cell lines:

  • RGC-5 cells (a retinal ganglion cell line) express OPA1 in mitochondria

  • Mixed retinal ganglion cell cultures show similar expression patterns to intact retina

These findings highlight the importance of OPA1 in tissues with high metabolic demands, particularly in the visual system.

How do researchers detect OPA1 in mitochondria using immunofluorescence?

For optimal detection of OPA1 in mitochondria using immunofluorescence, researchers employ the following techniques:

• Fixation for mitochondrial preservation:

  • Use 0.5% glutaraldehyde for 30 minutes at 4°C

  • This preserves mitochondrial morphology while maintaining protein antigenicity

• Co-localization with mitochondrial markers:

  • Perform double immunostaining with OPA1 antibody and anti-cytochrome c

  • This confirms mitochondrial localization of OPA1

• Signal amplification:

  • For weak signals, use Tyramide Signal Amplification Kit

  • This enhances detection sensitivity without increasing background

• Mitochondrial integrity verification:

  • Consider using additional mitochondrial markers that highlight different compartments

  • This helps distinguish between inner membrane, intermembrane space, and outer membrane localization

These approaches have successfully demonstrated OPA1's predominant localization in mitochondria of retinal ganglion cells, providing insights into its function in maintaining mitochondrial integrity in these cells.

What does western blot analysis reveal about OPA1 protein processing?

Western blot analysis has provided significant insights into OPA1 protein processing:

• Multiple isoform expression:

  • OPA1 antibodies typically detect two major bands in extracts of brain, retina, and optic nerve

  • These bands have approximate molecular weights of 90 and 80 kDa

  • A similar banding pattern is seen in mixed retinal ganglion cell cultures and RGC-5 cells

• Proteolytic processing:

  • The complex banding pattern of OPA1 is attributed to sequential proteolytic processing

  • Long forms can be cleaved to generate short forms with distinct functions

  • With optimized separation, up to five bands can be visualized in mouse samples

• Functional significance:

  • The long forms (top two bands) are associated with mitochondrial fusion

  • Short forms (bottom three bands) may have distinct roles in cristae structure

  • The balance between these forms is critical for normal mitochondrial function

• Tissue-specific patterns:

  • While the basic pattern is similar across tissues, subtle differences may exist

  • These differences may reflect tissue-specific regulation of OPA1 processing

Understanding these processing patterns is essential for interpreting experimental results and exploring how OPA1 dysfunction contributes to disease states.

How do OPA1 isoforms contribute to mitochondrial function?

OPA1 isoforms play distinct and complementary roles in mitochondrial function:

• Long forms:

  • Primarily involved in mitochondrial fusion

  • Anchored to the inner mitochondrial membrane

  • Essential for maintaining mitochondrial network connectivity

  • Help coordinate mitochondrial responses to cellular stress

• Short forms:

  • Generated through proteolytic processing of long forms

  • Thought to regulate cristae structure and organization

  • Important for maintaining efficient respiratory chain function

  • Contribute to mitochondrial DNA maintenance

• Balanced expression:

  • The ratio between long and short forms is tightly regulated

  • Disruption of this balance can affect mitochondrial morphology, energy production, and response to stress

  • Western blot analysis using optimized conditions can reveal shifts in this balance under various experimental conditions

These distinct functions highlight the importance of being able to detect and differentiate multiple OPA1 isoforms in research applications.

What advances have been made in OPA1 antibody techniques for studying disease models?

Recent advances in OPA1 antibody techniques have enhanced research in disease models:

• Improved western blot protocols:

  • Optimized conditions now allow visualization of up to five OPA1 isoforms

  • Extended run times at lower voltage significantly improve isoform separation

  • These improvements enable more detailed analysis of OPA1 processing in disease states

• Enhanced immunohistochemistry:

  • Better fixation methods preserve mitochondrial morphology while maintaining antigenicity

  • Signal amplification techniques improve detection sensitivity

  • These advances permit detailed analysis of OPA1 distribution in disease-affected tissues

• Specificity verification:

  • More rigorous controls for verifying antibody specificity

  • Pre-absorption with blocking peptides or recombinant OPA1

  • These methods ensure more reliable results in disease model research

These technical advances have facilitated more detailed investigations of OPA1's role in conditions such as optic atrophy and other mitochondrial disorders, potentially guiding future therapeutic approaches.