OPA1 Antibody, Biotin conjugated

What is OPA1 and what role does it play in mitochondrial function?

OPA1 (Optic Atrophy Protein 1) exists in two forms: long form (l-OPA1) anchored to the inner mitochondrial membrane and short form (s-OPA1) lacking the transmembrane anchor. These forms cooperate to regulate mitochondrial inner-membrane fusion . OPA1 functions as a dynamin-related GTPase critical for maintaining cristae structure and mitochondrial integrity. In brown adipose tissue, OPA1 deletion leads to mitochondrial dysfunction, characterized by reduced oxygen consumption and ATP synthesis rates, along with disrupted lamellar cristae structure and mitochondrial fragmentation .

What experimental applications is the OPA1 Antibody, Biotin conjugated suitable for?

The OPA1 Antibody, Biotin conjugated is validated for multiple applications including:

• Immunocytochemistry/Immunofluorescence (ICC/IF)

• Immunohistochemistry (IHC)

• Immunohistochemistry-Paraffin (IHC-P)

• Western Blot (WB)

The biotin conjugation enables detection via streptavidin systems, as demonstrated in validated protocols where cells were incubated with OPA1 Antibody conjugated to Biotin and subsequently detected with streptavidin conjugated to fluorophores like DyLight 550 .

What species reactivity does the OPA1 Antibody, Biotin conjugated demonstrate?

The OPA1 Antibody, Biotin conjugated demonstrates validated reactivity across multiple mammalian species:

• Human

• Mouse

• Rat

This cross-species reactivity makes it particularly valuable for comparative studies and translational research involving different model organisms.

What are the optimal conditions for using OPA1 Antibody, Biotin conjugated in immunofluorescence experiments?

For optimal immunofluorescence results, follow this validated protocol:

• Fix cells in 4% paraformaldehyde (10 minutes)

• Permeabilize using 0.05% Triton X-100 in PBS (5 minutes)

• Incubate with OPA1 Antibody conjugated to Biotin at 5 μg/ml (1 hour, room temperature)

• Detect using streptavidin conjugated to an appropriate fluorophore

• Counterstain nuclei with DAPI

While specific concentration requirements may vary by experimental conditions, starting dilutions of 1:50-1:200 are recommended for immunocytochemistry applications .

How should researchers validate the specificity of OPA1 Antibody, Biotin conjugated?

A comprehensive validation strategy includes:

What sample preparation techniques optimize detection of OPA1 in complex tissue samples?

For robust detection in tissue samples:

• For paraffin-embedded tissues, perform antigen retrieval appropriate for the tissue type

• Implement autofluorescence reduction strategies (e.g., Sudan Black B or copper sulfate treatment)

• Use fresh sections and optimize fixation parameters to preserve mitochondrial morphology

• Consider signal amplification methods such as tyramide signal amplification

• For tissues with high lipid content, ensure adequate permeabilization while preserving mitochondrial structures

How can researchers distinguish between long and short forms of OPA1 using the biotin-conjugated antibody?

Differentiating between l-OPA1 and s-OPA1 requires specialized approaches:

• Western blot analysis:

  • Use gradient gels (8-16%) to resolve the long (~100 kDa) and short (~80-90 kDa) forms

  • Include protease inhibitors during sample preparation to prevent artifactual processing

  • Use subcellular fractionation to separate membrane-bound l-OPA1 from soluble s-OPA1

• Quantitative assessment:

  • The ratio of s-OPA1 to l-OPA1 critically affects fusion efficiency, with an equimolar ratio showing optimal fusion capacity

  • An excess of s-OPA1 (2:1 ratio or greater) can disrupt l-OPA1:l-OPA1 homotypic interactions

• Co-localization studies:

  • Combine with super-resolution microscopy to visualize differential distribution patterns

  • Use membrane markers to distinguish membrane-anchored versus soluble populations

What strategies can researchers implement to investigate OPA1's role in mitochondrial membrane fusion?

Research has established that l-OPA1 mediates homotypic tethering of membranes containing cardiolipin, which is enhanced by GTP . To investigate fusion mechanisms:

• Membrane tethering assays:

  • Supported bilayer/liposome assays can demonstrate l-OPA1-mediated tethering

  • Homotypic l-OPA1:l-OPA1 interactions and heterotypic l-OPA1:cardiolipin interactions can be distinguished

  • GTP-dependence can be assessed using non-hydrolyzable analogues like GMPPCP

• Fusion efficiency analysis:

  • Quantify pore opening efficiency and dwell time before fusion

  • At equimolar levels of s-OPA1 and l-OPA1, fusion efficiency reaches 80% with a four-fold decrease in mean dwell time

  • Use mutations like G300E to disrupt GTP hydrolysis and analyze effects on tethering

• Lipid composition effects:

  • Cardiolipin is essential for l-OPA1 tethering enhancement by GTP

  • Control experiments should include membranes lacking cardiolipin (e.g., DOPC liposomes)

How can researchers quantitatively assess OPA1-dependent changes in mitochondrial morphology?

For quantitative analysis of mitochondrial morphology:

• Image acquisition protocols:

  • Obtain z-stacks with appropriate sampling to capture the full mitochondrial network

  • Use deconvolution to improve signal-to-noise ratio

  • Consider super-resolution approaches for improved morphological detail

• Morphometric analysis:

  • Measure parameters including aspect ratio, form factor, and branching index

  • Classify mitochondria as fragmented, intermediate, or tubular based on established criteria

  • Use software packages with batch processing capabilities for unbiased analysis

• Correlation with functional parameters:

  • Combine morphological analysis with measurements of membrane potential

  • Assess relationship between fragmentation and bioenergetic parameters

  • In OPA1-deficient brown adipose tissue, mitochondrial fragmentation correlates with impaired respiration and ATP synthesis

What are common technical challenges when using OPA1 Antibody, Biotin conjugated and how can they be resolved?

How should researchers interpret changes in OPA1 localization or expression patterns?

Interpreting OPA1 dynamics requires consideration of multiple factors:

• Context-dependent interpretation:

  • In brown adipose tissue, OPA1 deletion leads to mitochondrial fragmentation and cristae disruption

  • Changes in OPA1 expression should be correlated with functional outcomes like oxygen consumption rates

  • The ratio between long and short forms is as important as total OPA1 levels

• Experimental controls:

  • Include positive controls such as cells with known OPA1 dynamics

  • Use appropriate negative controls including antibody omission and isotype controls

  • Consider genetic controls like OPA1 knockout or knockdown models

• Integrated analysis:

  • Combine protein expression data with morphological assessment

  • Correlate with mitochondrial function parameters

  • Consider tissue-specific differences in OPA1 function and processing

What considerations are important for multiplex experiments including OPA1 Antibody, Biotin conjugated?

For successful multiplex experiments:

• Antibody compatibility planning:

  • Use primary antibodies from different species

  • For multiple biotin-conjugated antibodies, implement sequential detection with blocking steps

  • Validate antibodies individually before combining

• Detection strategy optimization:

  • Select fluorophores with minimal spectral overlap

  • Consider sequential rather than simultaneous detection

  • For streptavidin-based detection, use appropriate controls for endogenous biotin

• Co-localization studies:

  • Include established mitochondrial markers

  • Apply appropriate co-localization algorithms

  • Use high-resolution imaging techniques for accurate spatial analysis

How can researchers use OPA1 Antibody, Biotin conjugated to investigate the relationship between OPA1 and cardiolipin?

Research has shown that l-OPA1 can tether membranes through both homotypic l-OPA1:l-OPA1 interactions and heterotypic l-OPA1:cardiolipin interactions . To investigate this relationship:

• Membrane binding studies:

  • l-OPA1 can tether to cardiolipin-containing bilayers even without protein binding partners

  • This tethering is cardiolipin-dependent, as l-OPA1 does not tether to DOPC liposomes lacking cardiolipin

  • Use the antibody to track OPA1 localization in reconstituted systems

• Functional analysis:

  • GTP enhances l-OPA1-mediated tethering to cardiolipin-containing membranes

  • Use hydrolysis-dead mutants (G300E) to investigate the role of GTP hydrolysis

  • Combine with biophysical techniques to measure membrane binding affinities

• Visualization approaches:

  • Apply super-resolution microscopy to visualize OPA1-cardiolipin interactions

  • Use proximity ligation assays to detect close associations

  • Implement FRET-based approaches with labeled lipids

What experimental designs can help elucidate the cooperative functions of long and short OPA1 forms?

Research has shown that s-OPA1 and l-OPA1 cooperate to complete fusion of mitochondrial inner membranes . To investigate this cooperation:

• Ratio manipulation experiments:

  • l-OPA1-only homotypic fusion has low efficiency (~10%) and long dwell time (~20s)

  • Addition of s-OPA1 increases pore opening efficiency to 80% at equimolar ratio

  • Excessive s-OPA1 (ratio ≥2:1) disrupts tethering, suggesting a balanced ratio is critical

• Mutation studies:

  • The G300E mutation disrupts GTP hydrolysis and impairs tethering

  • Use site-directed mutagenesis to modify specific domains of each form

  • Analyze effects on fusion efficiency and kinetics

• Reconstitution approaches:

  • Use purified components to reconstitute fusion in vitro

  • Manipulate ratios of l-OPA1 and s-OPA1 to identify optimal conditions

  • Correlate with measurements from cellular systems

How can OPA1 Antibody, Biotin conjugated be utilized to study OPA1's role in metabolic regulation?

OPA1 deletion in brown adipose tissue (BAT) affects thermogenesis and metabolic regulation . To investigate these metabolic functions:

• Tissue-specific analysis:

  • Use the antibody to confirm OPA1 deletion in specific tissues (e.g., 10-fold reduction in BAT)

  • Verify that deletion is tissue-specific without affecting other tissues like WAT, liver, and skeletal muscle

  • Correlate with metabolic parameters like oxygen consumption

• Mitochondrial function assessment:

  • OPA1 deletion in BAT leads to impaired pyruvate-malate and palmitoyl-carnitine-dependent oxygen consumption

  • Correlate with ATP synthesis rates

  • Analyze relationship between mitochondrial morphology and function

• Structural-functional correlations:

  • OPA1 deletion causes "whitening" of BAT with increased numbers of enlarged unilocular lipid droplets

  • Mitochondria appear more fragmented with disrupted lamellar cristae

  • Use the antibody to track changes in OPA1 distribution during metabolic transitions