DPS Antibody

What is the DPS protein and why is it important in biological research?

DPS (decaprenyl diphosphate synthase subunit 1) is a 415-amino acid protein encoded by the PDSS1 gene in humans. This heterotetrameric enzyme plays a crucial role in the biosynthesis of ubiquinone (Coenzyme Q10) by catalyzing the condensation of farnesyl diphosphate (FPP) with isopentenyl diphosphate (IPP) to produce prenyl diphosphates of varying chain lengths . Its significance in research stems from its essential role in the mitochondrial respiratory chain, where ubiquinone serves as an electron carrier. DPS is also known by several synonyms including COQ1 and COQ10D2, with mutations in this gene linked to coenzyme Q10 deficiency syndromes . Understanding DPS function has implications for mitochondrial disorders, neurodegenerative diseases, and aging research.

What are the common applications of DPS antibodies in laboratory research?

DPS antibodies are primarily utilized for antigen-specific immunodetection in various biological samples . The most common applications include:

• Western Blotting: For specific detection and quantification of DPS protein in tissue or cell lysates, typically using reducing conditions and a primary anti-DPS antibody followed by a labeled secondary antibody

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of DPS in solution

• Immunohistochemistry/Immunocytochemistry: For localizing DPS within tissues or cells, confirming its mitochondrial localization

• Immunoprecipitation: For isolating DPS and associated protein complexes from biological samples

These techniques allow researchers to study DPS expression levels, localization patterns, and protein-protein interactions in various experimental conditions.

How should DPS antibodies be validated before experimental use?

Proper validation of DPS antibodies is essential to ensure experimental reliability and reproducibility. A comprehensive validation approach should include:

• Specificity testing:

  • Western blot analysis using positive controls (tissues/cells known to express DPS) and negative controls

  • Testing with recombinant DPS protein

  • Peptide competition assays to confirm binding to the target epitope

• Sensitivity assessment:

  • Determination of detection limits using serial dilutions of target protein

  • Comparison with other validated antibodies against the same target

• Cross-reactivity evaluation:

  • Testing against related proteins (particularly other COQ family members)

  • Species cross-reactivity assessment if using in different model organisms

• Application-specific validation:

  • For immunohistochemistry: confirmation of expected mitochondrial localization pattern

  • For flow cytometry: comparison with isotype controls

Each antibody should be validated specifically for the application and experimental system in which it will be used, as performance can vary significantly across different techniques .

What are the optimal conditions for using DPS antibodies in co-immunoprecipitation studies of mitochondrial protein complexes?

When designing co-immunoprecipitation (co-IP) experiments to study DPS interactions within mitochondrial protein complexes, several critical factors must be considered:

• Mitochondrial isolation and lysis:

  • Begin with differential centrifugation for crude mitochondrial isolation

  • Use gentle detergents (0.5-1% digitonin or 0.5% CHAPS) to preserve protein-protein interactions

  • Include protease inhibitors, phosphatase inhibitors, and reducing agents in all buffers

• Antibody selection and binding conditions:

  • Choose DPS antibodies raised against regions not involved in protein-protein interactions

  • Pre-clear lysates with appropriate control IgG and protein A/G beads

  • Optimize antibody concentration (typically 2-5 μg per 500 μg protein lysate)

  • Perform binding at 4°C overnight with gentle rotation

• Washing and elution strategies:

  • Use increasingly stringent washing buffers to remove non-specific interactions

  • Consider native elution with competing peptides for downstream functional assays

  • For MS analysis, on-bead digestion may preserve more interactions than elution

• Controls and validation:

  • Include isotype control antibodies

  • Use cells with DPS knockdown/knockout as negative controls

  • Confirm specific co-IP by reverse immunoprecipitation with antibodies against suspected interaction partners

This methodology has successfully identified interactions between DPS and other components of the coenzyme Q biosynthetic complex, including COQ2 and COQ4, providing insights into the regulation of ubiquinone biosynthesis .

How can epitope mapping be performed to characterize the binding specificity of different DPS antibodies?

Epitope mapping for DPS antibodies involves systematic characterization of the specific amino acid sequences recognized by the antibody. This process is critical for understanding antibody function and potential cross-reactivity. A comprehensive epitope mapping approach includes:

• Peptide array analysis:

  • Generate overlapping synthetic peptides (12-15 amino acids) spanning the entire DPS sequence

  • Immobilize peptides on cellulose membranes or glass slides

  • Probe with the DPS antibody followed by labeled secondary antibody

  • Identify positive signals corresponding to antibody binding regions

• Alanine scanning mutagenesis:

  • Once a general epitope region is identified, create peptides with systematic alanine substitutions

  • Analyze binding affinity changes to identify critical residues for antibody recognition

• Recombinant fragment approach:

  • Express different domains of DPS as recombinant fragments

  • Test antibody binding to identify domain-specific recognition

  • Further narrow down using overlapping peptides within positive domains

• Structural analysis:

  • If DPS crystal structure is available, map the identified epitope to understand its structural context

  • Assess accessibility of the epitope in native protein conformation

For example, some commercially available DPS antibodies have been developed against specific amino acid sequences such as MGKPT . Understanding the precise epitope helps researchers select appropriate antibodies for specific applications, particularly when studying different functional domains of the DPS protein or when distinguishing between closely related protein isoforms.

What strategies can overcome cross-reactivity issues when using DPS antibodies in tissues with high expression of related decaprenyl diphosphate synthase family members?

Cross-reactivity presents a significant challenge when studying DPS in tissues where related family members are expressed. Several methodological approaches can help overcome these issues:

• Antibody pre-absorption and competitive binding:

  • Pre-incubate DPS antibodies with recombinant related proteins

  • Use excess competing peptides from homologous regions to block cross-reactive binding

  • Verify specificity through western blotting against recombinant proteins of all family members

• Genetic approaches for specificity confirmation:

  • Use CRISPR/Cas9-mediated knockout/knockdown models as negative controls

  • Employ overexpression systems with tagged versions of DPS to differentiate from endogenous related proteins

  • Apply siRNA knockdown of specific family members to confirm antibody specificity

• Dual targeting strategies:

  • Use two different DPS antibodies targeting distinct epitopes

  • Consider proximity ligation assays requiring dual binding for signal generation

  • Employ co-localization with known DPS-interacting partners

• Advanced purification techniques:

  • Implement immunoaffinity chromatography with highly specific antibodies

  • Use two-dimensional electrophoresis to separate related proteins by both pI and molecular weight

  • Consider mass spectrometry validation of immunoprecipitated proteins

These techniques have successfully distinguished DPS (PDSS1) from related family members such as PDSS2, allowing for accurate characterization of specific protein functions without interference from homologous proteins .

How can DPS antibodies be effectively used to monitor changes in mitochondrial ubiquinone biosynthesis pathway in neurodegenerative disease models?

Leveraging DPS antibodies to monitor the ubiquinone biosynthesis pathway in neurodegenerative disease models requires a multifaceted approach:

• Quantitative assessment of DPS expression and localization:

  • Perform western blot analysis with standardized loading controls (VDAC or TOM20)

  • Use immunofluorescence with mitochondrial co-markers to assess changes in localization pattern

  • Apply super-resolution microscopy to visualize DPS distribution within mitochondrial subcompartments

• Analysis of DPS protein complexes:

  • Employ blue native PAGE followed by western blotting to analyze intact complexes

  • Perform co-immunoprecipitation to detect changes in interactions with other COQ proteins

  • Use proximity labeling techniques (BioID or APEX) coupled with DPS antibodies to identify altered interaction networks

• Functional correlation studies:

  • Correlate DPS protein levels/localization with ubiquinone levels (measured by HPLC)

  • Assess mitochondrial function parameters (oxygen consumption, membrane potential)

  • Measure downstream consequences of altered DPS function (ROS production, ATP levels)

• Intervention validation:

  • Use DPS antibodies to confirm target engagement after therapeutic interventions

  • Monitor restoration of normal DPS levels/localization after treatment

  • Track changes in DPS post-translational modifications that may regulate activity

This methodological framework has been valuable in identifying mitochondrial dysfunction in neurodegenerative conditions, where disruption of the ubiquinone biosynthesis pathway often precedes clinical manifestations. The approach allows researchers to determine whether alterations in DPS contribute to disease pathogenesis and whether targeting this pathway might offer therapeutic benefits .

What are the current techniques for developing highly specific monoclonal antibodies against different functional domains of the DPS protein?

The development of domain-specific DPS monoclonal antibodies involves sophisticated techniques to ensure both specificity and functionality:

• Strategic immunogen design:

  • Identify functional domains through bioinformatic analysis (e.g., catalytic site, FPP binding region)

  • Design peptides or recombinant fragments representing distinct domains

  • Consider both linear epitopes and conformation-dependent epitopes

  • Incorporate structural data to select accessible regions in the native protein

• Advanced immunization protocols:

  • Employ prime-boost strategies with different immunogen formulations

  • Use DNA immunization followed by protein boost to enhance response to conformational epitopes

  • Consider in vitro immunization with sequential epitope presentation

• High-throughput screening strategies:

  • Develop domain-specific ELISAs for initial screening

  • Implement functional assays to identify antibodies that modulate DPS activity

  • Use competition assays to identify antibodies targeting different epitopes

• Recombinant antibody engineering:

  • Apply phage display technology with synthetic or natural antibody libraries

  • Screen against multiple DPS domains simultaneously

  • Use deep sequencing of antibody repertoires to identify rare clones

• Validation in complex systems:

  • Test for function-blocking activity in cell-based assays

  • Verify domain specificity using truncated DPS variants

  • Confirm binding characteristics using surface plasmon resonance

This methodological approach enables the development of a panel of domain-specific antibodies that can be used to probe different aspects of DPS function, such as distinguishing between its roles in the initiation versus elongation phases of prenyl diphosphate synthesis .

What are the optimal fixation and antigen retrieval methods for immunohistochemical detection of DPS in different tissue types?

Optimizing immunohistochemical detection of DPS requires careful consideration of fixation and antigen retrieval methods based on tissue type and specific research questions:

• Fixation protocols by tissue type:

  Tissue Type	Recommended Fixation	Duration	Temperature
  Brain tissue	4% PFA	24-48h	4°C
  Muscle tissue	Acetone/methanol (1:1)	10-15 min	-20°C
  Liver tissue	10% neutral buffered formalin	24h	RT
  Cell cultures	4% PFA	15-20 min	RT

• Antigen retrieval methods and their effectiveness:

  Method	Conditions	Effectiveness for DPS	Considerations
  Heat-induced (citrate buffer, pH 6.0)	95-98°C, 20 min	High	Most widely applicable
  Heat-induced (EDTA buffer, pH 9.0)	95-98°C, 20 min	Very high	Better for formalin-fixed tissues
  Enzymatic (proteinase K)	37°C, 10-15 min	Moderate	Can damage tissue morphology
  No retrieval	N/A	Low	Only for acetone-fixed frozen sections

• Optimization strategies:

  • Perform titration experiments to determine optimal antibody concentration

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Compare signal-to-noise ratios across different retrieval methods

  • Consider dual immunofluorescence with mitochondrial markers for co-localization

• Special considerations:

  • For electron microscopy, use mild fixation (0.5-1% glutaraldehyde with 4% PFA)

  • For super-resolution microscopy, consider specialized tissue clearing techniques

  • When studying mitochondrial dynamics, rapid fixation is critical to preserve native state

These methodological considerations ensure maximum sensitivity while maintaining tissue morphology, allowing accurate assessment of DPS expression and localization across different experimental models .

How can researchers troubleshoot non-specific binding when using DPS antibodies in Western blotting applications?

Non-specific binding in Western blotting with DPS antibodies can significantly confound data interpretation. A systematic troubleshooting approach includes:

• Optimizing blocking conditions:

  • Test different blocking agents (5% non-fat milk, 5% BSA, commercial blockers)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions

• Primary antibody optimization:

  • Perform titration series (1:500 to 1:5000) to determine optimal concentration

  • Test different incubation temperatures (4°C, room temperature)

  • Add competing peptides to confirm specificity

  • Consider using alternative DPS antibodies targeting different epitopes

• Sample preparation refinements:

  • Ensure complete protein denaturation (heat at 95°C in reducing buffer)

  • Include additional protease inhibitors to prevent degradation

  • For mitochondrial proteins, consider specialized extraction methods

  • Use freshly prepared samples whenever possible

• Membrane washing procedures:

  • Increase number and duration of washes (5-6 washes of 10 minutes each)

  • Use higher concentrations of Tween-20 (0.1-0.5%) in wash buffer

  • Consider including 0.1% SDS in wash buffer for highly specific binding

• Detection system modifications:

  • Switch from chemiluminescence to fluorescence detection for better quantification

  • Use secondary antibodies with minimal cross-reactivity to sample species

  • Consider signal amplification systems only when target protein is in very low abundance

This methodical approach has successfully resolved non-specific binding issues in Western blotting applications, ensuring specific detection of DPS protein even in complex tissue lysates with abundant related proteins .

What are the most effective strategies for quantifying DPS protein levels in mitochondrial fractions from tissues with varying mitochondrial content?

Accurate quantification of DPS in tissues with varying mitochondrial content requires normalization strategies that account for these differences:

• Differential centrifugation and mitochondrial enrichment:

  • Implement step-wise centrifugation protocol (600g → 3,000g → 10,000g)

  • Purify mitochondria using density gradient centrifugation

  • Assess purity by Western blotting for markers of other cellular compartments

• Normalization strategies:

  Normalization Method	Advantages	Limitations	Best Application
  Total protein	Simple, universal	Doesn't account for mitochondrial content	Preliminary studies
  Mitochondrial markers (VDAC, TOM20)	Accounts for mitochondrial mass	Marker may vary independently	Comparing similar tissue types
  Mitochondrial DNA	Accurate measure of mitochondrial number	Requires separate DNA extraction	Tissues with dramatic differences
  Multiple mitochondrial proteins	Robust to individual protein variations	Labor intensive	High-precision studies
  Citrate synthase activity	Functional measure of mitochondrial mass	Requires additional enzymatic assay	Gold standard for functional studies

• Quantification methods:

  • Fluorescence-based Western blotting with internal standards

  • ELISA using recombinant DPS protein standards

  • Mass spectrometry with stable isotope-labeled peptide standards

  • Capillary Western immunoassay (Wes) for small sample volumes

• Data interpretation considerations:

  • Calculate DPS/mitochondrial marker ratios

  • Consider multiple normalization approaches and compare results

  • Account for tissue-specific differences in mitochondrial composition

  • Include quality controls with known DPS expression levels

This comprehensive approach allows for accurate quantification of DPS protein levels across different tissues, enabling meaningful comparisons in various physiological and pathological conditions .

How can DPS antibodies be integrated into high-throughput screening approaches for identifying compounds that modulate ubiquinone biosynthesis?

Integrating DPS antibodies into high-throughput screening (HTS) methodologies creates powerful platforms for identifying modulators of ubiquinone biosynthesis:

• Cell-based screening systems:

  • Develop stable cell lines expressing fluorescently-tagged DPS

  • Create reporter systems where DPS levels or localization is linked to fluorescent output

  • Implement automated microscopy to detect changes in DPS expression/localization after compound treatment

• Antibody-based detection platforms:

  • Adapt AlphaLISA or homogeneous time-resolved fluorescence (HTRF) assays using DPS antibodies

  • Develop high-content imaging assays to monitor DPS co-localization with other pathway components

  • Design split-luciferase complementation assays to monitor DPS protein interactions

• Functional screening approaches:

  • Couple DPS detection with downstream ubiquinone quantification

  • Measure mitochondrial function parameters (membrane potential, oxygen consumption)

  • Develop FRET-based biosensors for DPS enzymatic activity

• Data analysis and validation:

  • Implement machine learning algorithms to identify subtle phenotypic changes

  • Cluster compounds by mechanism based on multiparametric analysis

  • Validate hits through orthogonal assays including enzyme activity measurements

This methodological framework allows for the screening of thousands of compounds to identify those that specifically modulate DPS expression, localization, or function, potentially leading to therapeutic candidates for mitochondrial disorders associated with ubiquinone deficiency .

What approaches can be used to study post-translational modifications of DPS protein using specific antibodies?

Studying post-translational modifications (PTMs) of DPS requires specialized antibody-based approaches:

• Modification-specific antibody development:

  • Generate antibodies against known or predicted PTM sites (phosphorylation, acetylation, etc.)

  • Develop antibodies specific to modified forms using synthetic peptides with the modification

  • Validate specificity against modified and unmodified recombinant protein

• Enrichment strategies for modified DPS:

  • Immunoprecipitation with pan-DPS antibodies followed by PTM-specific detection

  • Tandem affinity purification using antibodies against both DPS and the modification

  • PTM-specific enrichment (e.g., phosphopeptide enrichment) followed by DPS detection

• Detection and quantification methods:

  Method	Applications	Sensitivity	Specificity
  Western blot with PTM-specific antibodies	Relative quantification	Medium	Medium-High
  Mass spectrometry after IP	Site identification and quantification	Very High	Very High
  Proximity ligation assay	In situ detection	High	High
  Phos-tag SDS-PAGE	Phosphorylation detection	Medium	Medium

• Functional correlation approaches:

  • Correlation of PTM status with enzyme activity

  • Study of modification dynamics during cellular stress

  • Identification of enzymes responsible for adding/removing modifications

  • Creation of modification-mimetic mutants for functional studies

This comprehensive approach allows researchers to identify which PTMs regulate DPS activity, localization, and protein-protein interactions, providing insights into the regulation of ubiquinone biosynthesis under different physiological conditions .

How can researchers design experiments to investigate the role of DPS in mitochondrial dysfunction using antibody-based techniques?

Designing experiments to investigate DPS's role in mitochondrial dysfunction requires a multi-faceted approach:

• Expression and localization analysis across disease models:

  • Compare DPS levels in affected vs. unaffected tissues using Western blotting

  • Analyze subcellular distribution using immunofluorescence and super-resolution microscopy

  • Perform temporal studies to determine if DPS alterations precede other mitochondrial defects

• Protein interaction network analysis:

  • Map DPS interactome using immunoprecipitation coupled with mass spectrometry

  • Analyze changes in protein interactions during mitochondrial stress

  • Employ proximity labeling techniques to identify transient interactions

• Genetic manipulation combined with antibody detection:

  • Perform siRNA knockdown or CRISPR/Cas9 knockout of DPS

  • Rescue experiments with wild-type vs. mutant DPS

  • Create cell lines with inducible expression for temporal studies

• Functional correlation studies:

  • Correlate DPS levels/localization with:

    • Ubiquinone content (HPLC analysis)

    • Mitochondrial respiration (Seahorse analysis)

    • ROS production (fluorescent probes)

    • Mitochondrial morphology (electron microscopy)

• Intervention validation:

  • Test compounds that stabilize DPS or enhance its activity

  • Examine effects of ubiquinone supplementation on DPS expression/localization

  • Use gene therapy approaches to restore normal DPS levels

This experimental framework enables researchers to establish causal relationships between DPS dysfunction and mitochondrial pathology, potentially identifying new therapeutic targets for mitochondrial disorders .