AASDHPPT Antibody

What is AASDHPPT and what is its primary function in cellular metabolism?

AASDHPPT (L-aminoadipate-semialdehyde dehydrogenase-phosphopantetheinyl transferase) is an enzyme that catalyzes critical post-translational modifications by transferring the 4'-phosphopantetheine moiety from coenzyme A to serine residues on target proteins. It plays an essential role in modifying various acceptor proteins, including the acyl carrier domain of fatty acid synthase (FASN) .

The enzyme is required for mitochondrial respiration and oxidative metabolism via the mitochondrial fatty acid synthesis (mtFAS) pathway. AASDHPPT can transfer phosphopantetheine regardless of whether CoA is presented in the free thiol form or as an acetyl thioester . The protein is similar to Saccharomyces cerevisiae LYS5, which activates alpha-aminoadipate dehydrogenase in the lysine biosynthetic pathway, and there's evidence suggesting that defects in the human gene might result in pipecolic acidemia .

What immunogen strategies are most effective for AASDHPPT antibody production?

Based on commercial antibody data, several immunogen strategies have proven effective for generating AASDHPPT antibodies:

For optimal specificity, epitope-specific immunogen approaches targeting amino acids 11-60 have shown broad application compatibility across multiple species (human, mouse, rat) .

What is the molecular weight and cellular localization of AASDHPPT?

AASDHPPT has a calculated molecular weight of approximately 35-36 kDa, which is consistently observed in Western blot applications . The protein localizes primarily to the cytoplasm and cytosol according to subcellular localization studies .

The Human Protein Atlas project has conducted extensive validation through immunohistochemistry and immunofluorescence, providing detailed spatial information about AASDHPPT expression across hundreds of normal and disease tissues, as well as at the subcellular level .

What are the recommended dilution ranges for different experimental applications of AASDHPPT antibodies?

The optimal dilution ranges vary by application technique and specific antibody clone:

It is strongly recommended to titrate each antibody in your specific experimental system as sample type, fixation method, and detection system can all influence optimal antibody concentration .

How should researchers validate AASDHPPT antibody specificity in their experimental systems?

A comprehensive validation approach should include:

• Positive and negative controls: Use tissues/cells known to express or lack AASDHPPT (human brain tissue has been validated as a positive control for Western blot)

• Multiple antibody comparison: Employ antibodies targeting different epitopes of AASDHPPT (N-terminal vs. C-terminal) to confirm consistent staining patterns

• Protein array validation: Some commercial antibodies have been tested against protein arrays containing 364 human recombinant protein fragments to ensure specificity

• Molecular weight verification: Confirm the observed band matches the predicted 35-36 kDa size of AASDHPPT

• Knockdown/knockout controls: When possible, use siRNA silencing of AASDHPPT to demonstrate antibody specificity, as studies have shown siRNA silencing completely blocks the post-translational modification function of AASDHPPT

Prestige Antibodies from the Human Protein Atlas undergo particularly extensive validation including IHC tissue arrays of 44 normal human tissues and 20 common cancer types, providing an excellent benchmark for specificity .

What sample preparation protocols optimize AASDHPPT detection in different applications?

For optimal detection of AASDHPPT across applications:

Western Blot:

• Use fresh lysates in RIPA buffer containing protease inhibitors

• Denature proteins at 95°C for 5 minutes in reducing conditions

• Load 20-50 μg of total protein per lane

• Transfer to PVDF membrane (preferred over nitrocellulose for this protein)

• HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate has been verified as a reliable positive control

Immunohistochemistry:

• Paraffin-embedded sections (4-6 μm thick) with antigen retrieval

• Citrate buffer (pH 6.0) heat-induced epitope retrieval has shown good results

• Human brain tissue sections have been successfully used for validation

• Block with 5% normal serum from the same species as the secondary antibody

Immunofluorescence:

• HeLa cells serve as a reliable positive control

• 4% paraformaldehyde fixation for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 10 minutes

• Block with 3% BSA in PBS for 1 hour at room temperature

How is AASDHPPT expression altered in neurological disorders and what methodological approaches can detect these changes?

Research has implicated AASDHPPT in neurological disorders, particularly in Neurodegeneration with Brain Iron Accumulation (NBIA) disorders like PKAN (Pantothenate Kinase-Associated Neurodegeneration). In PKAN fibroblasts, the expression levels of AASDHPPT were markedly increased compared to control cells .

Methodological Approaches:

• Quantitative Western Blot: Use standardized loading controls (GAPDH or β-actin) and densitometry analysis to quantify expression differences between patient and control samples

• qRT-PCR: Monitor changes in AASDHPPT transcript levels, as studies have shown correlation between protein and mRNA levels in treatment responses

• Treatment Response Analysis: Pantothenate treatment has been shown to correct AASDHPPT expression levels in specific PKAN mutations (P1 and P2) but not in fibroblasts harboring bi-allelic mutations encoding truncated PANK2 proteins (P3)

• Dose-Dependent Studies: Design experiments with increasing concentrations of therapeutic compounds (like pantothenate) to determine critical thresholds for normalizing AASDHPPT expression

For analyzing PKAN patient samples specifically, researchers should consider the mutation type, as different PANK2 mutations respond differently to treatment interventions .

What role does AASDHPPT play in mitochondrial fatty acid synthesis, and how can researchers study this function?

AASDHPPT is crucial for the mitochondrial fatty acid synthesis (mtFAS) pathway, which is essential for mitochondrial respiration and oxidative metabolism. The enzyme is required for the phosphopantetheinylation of mitochondrial acyl carrier protein (mtACP) .

Research Methodologies:

• Co-immunoprecipitation: Use AASDHPPT antibodies to pull down protein complexes and identify interacting partners in the mtFAS pathway

• Proximity Ligation Assay: Detect in situ interactions between AASDHPPT and its mitochondrial targets

• Phosphopantetheinylation Assays: Monitor the transfer of 4'-phosphopantetheine using radiolabeled CoA substrates or specific antibodies against phosphopantetheinylated forms of target proteins

• Mitochondrial Fractionation: Isolate mitochondria to study localized AASDHPPT activity separate from cytosolic functions

• Respiratory Chain Analysis: Measure oxygen consumption rate (OCR) in cells with manipulated AASDHPPT levels to assess impact on mitochondrial respiratory function

Studies have shown that siRNA silencing of AASDHPPT completely blocks the post-translational modification of 10-FTHFDH (10-formyltetrahydrofolate dehydrogenase), which requires a 4'-phosphopantetheine cofactor for catalysis. A mitochondrial homolog of 10-FTHFDH is activated by the same AASDHPPT enzyme .

How can researchers use AASDHPPT antibodies to investigate cellular responses to metabolic stress?

AASDHPPT plays a critical role in metabolic pathways, and its regulation may serve as a marker for cellular adaptations to stress conditions:

Experimental Approaches:

• Stress Induction Models: Subject cells to oxidative stress, nutrient deprivation, or hypoxia and monitor AASDHPPT expression and localization changes using immunofluorescence and Western blot

• Metabolic Flux Analysis: Combine AASDHPPT antibody-based detection with stable isotope tracing to correlate enzyme expression with metabolic pathway activity

• Time-Course Studies: Monitor dynamic changes in AASDHPPT levels during acute and chronic stress responses using standardized antibody-based quantification

• Compartment-Specific Analysis: Use subcellular fractionation combined with AASDHPPT immunoblotting to detect translocation between cellular compartments under stress conditions

• Co-Expression Analysis: Perform multiplex immunofluorescence to simultaneously detect AASDHPPT and stress markers or interacting partners

In PKAN cells with low mitochondrial CoA levels, AASDHPPT expression levels were markedly increased, suggesting a compensatory mechanism. This upregulation could be reversed in specific mutations through pantothenate treatment, indicating that AASDHPPT expression is responsive to metabolic interventions .

What techniques can be used to study AASDHPPT's role in protein post-translational modifications?

AASDHPPT's primary function involves post-translational phosphopantetheinylation of target proteins. Several techniques can be employed to study this activity:

• Mass Spectrometry: Identify phosphopantetheinylated proteins and quantify modification stoichiometry before and after AASDHPPT manipulation

• Recombinant Protein Assays: Express and purify AASDHPPT for in vitro phosphopantetheinylation assays with known and potential substrates

• Antibody-Based Detection: Develop modification-specific antibodies that recognize the phosphopantetheine moiety on target proteins

• Protein Domain Swapping: Create chimeric proteins with or without the serine residue targeted by AASDHPPT to analyze functional consequences of modification

• Structural Biology Approaches: Leverage the available crystal structure of human AASDHPPT to design experiments probing the molecular mechanism of the enzyme

The human PPTase AASDHPPT has been demonstrated to act on several apo-proteins, suggesting it is not specific for particular proteins but rather recognizes a structural motif. This broad substrate recognition makes it an interesting target for studying post-translational regulation of multiple pathways simultaneously .

What are common challenges when detecting AASDHPPT in Western blot applications?

Researchers may encounter several technical challenges when performing Western blots for AASDHPPT:

• Non-specific bands: Due to the relatively small size of AASDHPPT (35-36 kDa), distinguish it from similar-sized proteins by:

  • Using gradient gels (10-15%) for better resolution in this range

  • Including positive control lysates (HepG2 or human brain tissue)

  • Comparing results with antibodies targeting different epitopes

• Weak signal: Optimize detection by:

  • Reducing antibody dilution (try 1:200-1:500 range initially)

  • Extending primary antibody incubation to overnight at 4°C

  • Using enhanced chemiluminescence (ECL) substrates specifically designed for low-abundance proteins

• Background issues: Minimize by:

  • Extending blocking time (2 hours at room temperature or overnight at 4°C)

  • Using 5% BSA instead of milk for blocking when phospho-specific detection is important

  • Increasing wash steps (5 x 5 minutes) with 0.1% Tween-20 in PBS

• Inconsistent loading: Ensure reliable quantification by:

  • Confirming equal loading with total protein stains (Ponceau S or SYPRO Ruby)

  • Using validated housekeeping proteins as loading controls (β-actin or GAPDH)

  • Performing technical replicates across multiple blots

How can immunohistochemistry protocols be optimized for AASDHPPT detection in different tissue types?

Optimizing IHC for AASDHPPT requires tissue-specific considerations:

• Fixation methods:

  • For brain tissue: 10% neutral buffered formalin for 24-48 hours is typically sufficient

  • For high-lipid tissues: Extended fixation times may be necessary for adequate penetration

  • Fresh-frozen sections may yield greater sensitivity for certain applications

• Antigen retrieval optimization:

  • Test multiple methods: citrate buffer (pH 6.0), EDTA buffer (pH 8.0), or enzymatic retrieval

  • Adjust retrieval time: 10-20 minutes for pressure cooker methods, 30-40 minutes for water bath methods

• Blocking strategies:

  • For high background tissues: Add 0.1-0.3% Triton X-100 to blocking solution

  • For lipid-rich tissues: Consider pre-treatment with 0.3% hydrogen peroxide to reduce endogenous peroxidase activity

• Detection system selection:

  • For low expression: Use polymer-based detection systems or tyramide signal amplification

  • For co-localization studies: Consider fluorescent secondaries with specific filters to minimize spectral overlap

• Antibody validation across tissues:

  • Positive control: Human brain tissue has been validated for AASDHPPT detection

  • Titration ranges: Test dilutions between 1:50-1:300 for optimal signal-to-noise ratio

The Human Protein Atlas provides extensive validation data across 44 normal human tissues, which can serve as an excellent reference for expected staining patterns .

What are the recommended controls for validating AASDHPPT antibody specificity and sensitivity?

A comprehensive control strategy should include:

• Positive and negative tissue controls:

  • Positive: Human brain tissue and HepG2 cell lysates have confirmed AASDHPPT expression

  • Negative: Tissues with minimal expression or cells with AASDHPPT knockdown

• Antibody controls:

  • Primary antibody omission: To assess secondary antibody non-specific binding

  • Isotype control: IgG from the same species at equivalent concentration

  • Blocking peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

• Genetic validation:

  • siRNA knockdown: Confirm decreased signal with AASDHPPT-targeted siRNA

  • Overexpression systems: Verify increased signal in transfected cells

• Multiple antibody comparison:

  • Use antibodies targeting different epitopes (N-terminal vs. C-terminal)

  • Compare polyclonal and monoclonal antibodies when available

• Cross-species validation:

  • Test antibody in multiple species with known sequence homology

  • Human AASDHPPT shows high sequence conservation with mouse (92%) and rat (92%) , making these viable cross-species controls

• Recombinant protein controls:

  • Use purified recombinant AASDHPPT as a positive control in immunoblotting

  • Create standard curves with known quantities for quantitative applications