PRODH Antibody

What is PRODH and why is it important in cellular metabolism?

PRODH (Proline Dehydrogenase) is a 516 amino acid protein that plays a crucial role in cellular metabolism by catalyzing the first step in proline degradation, converting proline to delta-1-pyrroline-5-carboxylate. This enzymatic process is particularly significant as it is induced during p53-mediated apoptosis, creating an important link between PRODH and cellular stress responses and programmed cell death mechanisms . The enzyme's activity contributes to energy metabolism and potentially to cellular redox regulation, making it an important target for studies focusing on metabolic disorders and cancer research.

Where is PRODH primarily localized in cells?

PRODH is primarily localized in the mitochondrial matrix, which aligns with its metabolic function in amino acid catabolism . This subcellular localization has been confirmed through multiple experimental approaches, including digitonin extraction of intact cells and immunofluorescence microscopy . When performing subcellular fractionation experiments, PRODH activity co-localizes with citrate synthase (a mitochondrial marker) rather than with cytosolic or glycosomal markers . Western blot analysis of purified mitochondrial vesicles from epimastigote stage cells reveals a single band with an apparent molecular mass of 140 kDa, confirming PRODH as a mitochondrial membrane-located, FAD-dependent enzyme .

What are the most effective applications for PRODH antibodies?

PRODH antibodies are effective in multiple laboratory applications, with the most reliable being western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) . When selecting a PRODH antibody, it's important to verify that it has been validated for your specific application of interest. Monoclonal antibodies like the A-11 mouse monoclonal IgG1 kappa light chain antibody offer high specificity for detecting PRODH protein of human origin across these applications . For optimal results in immunofluorescence studies, combining PRODH antibody detection with mitochondrial markers (such as MitoTracker) can provide confirmatory evidence of proper localization and antibody specificity.

What methods can be used to validate PRODH antibody specificity for research applications?

Validating PRODH antibody specificity requires a multi-faceted approach:

• Western blot analysis with competing peptides: Perform competition assays by incubating nitrocellulose membranes with synthetic peptides before introducing the anti-PRODH antibody. A significant reduction in signal indicates antibody specificity to the target epitope .

• Subcellular fractionation correlation: Compare PRODH antibody detection patterns with known mitochondrial markers in fractionation experiments. PRODH signals should correlate with mitochondrial fraction markers like citrate synthase rather than cytosolic (pyruvate kinase) or glycosomal (hexokinase) markers .

• Immunofluorescence co-localization: Confirm specificity through co-localization studies using DAPI for DNA staining (blue) and MitoTracker Red for mitochondrial staining, alongside the PRODH antibody coupled to fluorescent probes (e.g., AlexaFluor-455) .

• Published validation data review: Examine the antibody's validation history in peer-reviewed publications, specifically looking for consistent detection patterns across different experimental systems and conditions .

How can researchers accurately measure PRODH enzyme activity in conjunction with antibody-based detection methods?

PRODH enzyme activity can be measured using two complementary approaches while correlating with antibody-based detection:

• DCPIP reduction assay: Measure the reduction of the electron-accepting dye dichlorophenolindophenol (DCPIP) at 600 nm. The reaction mixture should contain 11 mM MOPS, 11 mM MgCl₂, 11% (v/v) glycerol, 0.28 mM phenazine methosulfate, and 56 μM of DCPIP at pH 7.5. Add varying proline concentrations to the assay mix and initiate the reaction by adding the enzyme. Calculate activity using an absorption coefficient (ε) of 21 mM⁻¹·cm⁻¹ at 600 nm for DCPIP .

• FAD-linked enzyme quantification: Determine the concentration of flavin-bound PRODH using the molar extinction coefficient for bound FAD (ε₄₅₁ = 10,800 M⁻¹·cm⁻¹) . This approach allows for specific quantification of the active enzyme form.

• Correlation with antibody detection: After measuring enzyme activity, perform western blotting on the same samples to correlate protein expression levels with enzymatic activity. This combined approach provides insights into both the presence and functional state of PRODH .

What are the critical considerations when studying PRODH isoforms using antibodies?

When studying PRODH isoforms, researchers should consider:

• Isoform specificity: PRODH exists as two isoforms generated through alternative splicing, which may have distinct functional implications in various tissues . Verify whether your antibody recognizes epitopes common to both isoforms or is specific to one variant.

• Tissue expression patterns: PRODH shows predominant expression in lung, brain, and skeletal muscle, with lower expression levels in heart, liver, kidney, and pancreas . Design experiments that account for these tissue-specific expression patterns.

• Cross-reactivity assessment: Test the antibody against recombinant versions of both isoforms to determine specificity and potential cross-reactivity.

• Epitope mapping: Understand the exact epitope recognized by the antibody and its preservation across isoforms, species, and experimental conditions.

• Control samples: Include appropriate positive and negative controls, including tissues or cells known to express specific isoforms at varying levels.

How should researchers design experiments to study PRODH's role in p53-mediated apoptosis?

To effectively study PRODH's role in p53-mediated apoptosis:

• Establish appropriate cellular models: Select cell lines with functional p53 and create paired controls with p53 knockdown/knockout to distinguish p53-dependent effects.

• Induce p53 activation: Use genotoxic agents (e.g., doxorubicin, cisplatin) or non-genotoxic p53 activators (e.g., Nutlin-3a) at various concentrations and time points.

• Monitor PRODH expression and activity:

  • Perform western blotting with PRODH antibodies to track protein expression changes

  • Measure PRODH enzymatic activity using the DCPIP reduction assay

  • Use immunofluorescence to monitor subcellular localization changes

• Assess apoptotic markers concurrently: Measure caspase activation, PARP cleavage, and phosphatidylserine externalization to correlate with PRODH changes.

• Manipulate PRODH levels: Use siRNA/shRNA knockdown or CRISPR-Cas9 editing to reduce PRODH expression, or overexpression systems to increase it, then observe effects on apoptotic response.

• Analyze proline metabolism: Measure proline levels and P5C (pyrroline-5-carboxylate) production to connect enzymatic activity with downstream metabolic effects.

• Evaluate ROS production: Since PRODH activity may influence reactive oxygen species generation, include oxidative stress measurements using fluorescent probes.

What are the best practices for troubleshooting non-specific binding when using PRODH antibodies in immunohistochemistry?

When troubleshooting non-specific binding in PRODH immunohistochemistry:

• Optimize blocking conditions: Test different blocking agents (BSA, normal serum, commercial blockers) at various concentrations (3-5%) and incubation times (1-2 hours).

• Titrate antibody concentration: Perform a dilution series of the primary antibody to determine the optimal concentration that maximizes specific signal while minimizing background.

• Include peptide competition controls: Pre-incubate the antibody with synthetic peptide corresponding to the target epitope to verify signal specificity .

• Use appropriate negative controls:

  • Tissue sections known not to express PRODH

  • Primary antibody omission

  • Isotype control antibodies at the same concentration

• Modify antigen retrieval methods: Compare heat-induced epitope retrieval using different buffers (citrate, EDTA, Tris) and enzymatic retrieval approaches.

• Adjust washing protocols: Increase wash stringency with higher salt concentrations or longer/more frequent washing steps.

• Validate antibody quality: Ensure you're using antibodies from reputable sources with demonstrated validation data for immunohistochemistry applications .

• Compare multiple detection systems: Test different secondary antibodies and visualization methods (fluorescent vs. chromogenic) to identify optimal detection conditions.

How can PRODH antibodies be used to investigate its role in hyperprolinemia and associated neuropsychiatric disorders?

Investigating PRODH's role in hyperprolinemia and neuropsychiatric disorders requires:

• Patient sample analysis:

  • Use western blotting with PRODH antibodies to compare expression levels in accessible tissues (blood cells, skin fibroblasts) from patients with hyperprolinemia type 1 versus controls

  • Apply immunohistochemistry on post-mortem brain tissues to examine regional expression patterns in relevant neuropsychiatric conditions

• Functional variant characterization:

  • Develop site-specific antibodies that can distinguish wild-type PRODH from disease-associated variants

  • Express recombinant wild-type and mutant PRODH proteins and compare antibody reactivity patterns and enzyme activity correlations

• Model system development:

  • Generate cellular and animal models of PRODH deficiency

  • Apply immunofluorescence techniques to study subcellular localization changes in disease models

  • Combine with biochemical assays to correlate protein expression with functional deficits

• Clinical correlations:

  • Use validated PRODH antibodies to measure expression levels in patient-derived samples

  • Correlate protein levels with clinical parameters such as serum proline levels, severity of psychiatric symptoms, and neuroimaging findings

• Potential biomarker evaluation:

  • Assess whether PRODH protein levels or post-translational modifications detected by specific antibodies correlate with disease progression or treatment response

What considerations are important when selecting PRODH antibodies for studying its potential role in cancer metabolism?

When studying PRODH in cancer metabolism, consider:

• Epitope accessibility in cancer tissues: Select antibodies targeting epitopes that remain accessible in the context of cancer-associated post-translational modifications or in protein complexes.

• Validation in relevant cancer models: Prioritize antibodies with demonstrated specificity in cancer cell lines and tumor tissues relevant to your research focus .

• Compatibility with multiplexed approaches: Choose antibodies compatible with multi-parameter analyses to simultaneously examine PRODH alongside other metabolism-related proteins.

• Context-specific validation:

  • Test antibody performance under conditions that mimic the tumor microenvironment (hypoxia, nutrient deprivation)

  • Validate specificity in both normoxic and hypoxic conditions, as metabolic enzyme expression and localization may change

• Isoform specificity relevance: Determine whether cancer tissues express specific PRODH isoforms and select antibodies accordingly .

• Sensitivity considerations: For detection of potentially low-abundance PRODH in certain cancers, select antibodies with demonstrated high sensitivity and signal-to-noise ratio.

• Application flexibility: Select antibodies validated across multiple applications (WB, IHC, IF) to enable comprehensive analysis of both expression and localization .

What are the optimal storage and handling conditions for maintaining PRODH antibody performance over time?

To maintain optimal PRODH antibody performance:

• Storage temperature:

  • Store unconjugated antibodies at -20°C for long-term storage

  • Store working aliquots at 4°C for up to one month

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Buffer considerations:

  • Ensure storage buffers contain appropriate preservatives (0.02-0.05% sodium azide)

  • For conjugated antibodies (HRP, fluorophores), follow manufacturer recommendations for light protection and special buffer requirements

• Stabilizing additives:

  • Consider adding protein stabilizers (BSA, glycerol) for diluted antibody preparations

  • Typical working solutions contain 1% BSA and up to 50% glycerol

• Quality control monitoring:

  • Periodically test antibody performance using consistent positive control samples

  • Document lot-to-lot variation by testing new lots against reference standards

• Transport conditions:

  • Transport on ice or with cold packs

  • Ensure temperature logging for valuable or sensitive antibody preparations

• Contamination prevention:

  • Use sterile technique when handling antibody solutions

  • Filter solutions if necessary to remove particulates

• Record keeping:

  • Maintain detailed logs of antibody handling, including freeze-thaw cycles, dilution dates, and performance observations

  • Record lot numbers and purchase dates to track performance over time

How can researchers effectively validate PRODH antibodies across different species for comparative studies?

For cross-species PRODH antibody validation:

• Sequence alignment analysis:

  • Compare PRODH protein sequences across target species

  • Identify conserved and variable regions, focusing on epitope conservation

  • Select antibodies raised against highly conserved epitopes for multi-species applications

• Graduated validation approach:

  • Begin with western blotting to confirm detection of appropriately sized bands across species

  • Proceed to immunoprecipitation to verify target specificity

  • Finally validate for immunohistochemistry/immunofluorescence applications

• Species-specific controls:

  • Use PRODH-knockout or knockdown samples from each species when available

  • Include recombinant PRODH proteins from each species as positive controls

  • Employ peptide competition assays using species-specific peptides

• Tissue/subcellular localization confirmation:

  • Verify that detected PRODH localizes to mitochondria across all species

  • Compare expression patterns across tissues known to express PRODH (lung, brain, skeletal muscle)

• Functional correlation:

  • Correlate antibody detection with enzymatic activity measurements across species

  • Ensure that enzymatic activity matches protein levels detected by the antibody

• Specificity confirmation:

  • Test for cross-reactivity with related enzymes (P5C dehydrogenase, P5C reductase)

  • Evaluate potential cross-reactivity with species-specific protein homologs