Customize PAP7 Antibody

Description

PAP7 Antibody Development and Specificity

PAP7 (PBR-associated protein 7) is a 52-kDa protein identified through yeast two-hybrid screenings using the peripheral-type benzodiazepine receptor (PBR) and PKA regulatory subunit RIα as bait . The anti-PAP7 antibody was generated using a synthetic peptide antigen corresponding to a unique region of PAP7. Key validation steps include:

Immunoblotting: Detected a single 52-kDa band in MA-10 Leydig cells, PC12 cells transfected with PAP7 cDNA, and human tissues .
Immunocytochemistry: Localized PAP7 to the cytoplasm in MA-10 cells, with signal neutralization by preabsorption with the antigenic peptide .
Immunoprecipitation: Co-precipitated PKA-RIα from human testis lysates, confirming in vivo interactions .

Role in Steroidogenesis

PAP7 facilitates cholesterol transport into mitochondria by linking PKA-RIα to PBR-rich organelles. Key findings using the antibody include:

Experimental Model	Key Observation	Citation
MA-10 Leydig cells	PAP7 overexpression increased hCG-induced steroidogenesis; partial PAP7 inhibited it
Antisense oligonucleotide treatment	Reduced hCG-stimulated steroid production by 50–70%
Human testis tissue	Co-immunoprecipitation confirmed PAP7-PKA-RIα interaction

Mechanistic Studies

In vitro binding assays: GST-PAP7 fusion proteins pulled down PBR (mitochondrial) and PKA-RIα (cytosolic) .
Subcellular localization: PAP7 antibody revealed cytoplasmic distribution, consistent with its role in PKA targeting .

Beyond Steroidogenesis: PAP7 in Neuronal and Iron Regulation

PAP7 antibody has also been utilized in neurobiological contexts:

Mouse brain lysates: Immunoprecipitation demonstrated PAP7 interaction with Dexras1 (a GTPase) and DMT1 (iron transporter), implicating it in neuronal iron homeostasis .
PC12 cells: Endogenous PAP7-Dexras1 complexes were identified, suggesting cross-talk between steroidogenic and iron-regulatory pathways .

Technical Considerations

Antibody specificity: No cross-reactivity observed with unrelated proteins in control experiments .
Limitations: Weak interaction with PKA-RIIα noted in vitro but not in yeast two-hybrid systems .

Implications for Therapeutic Research

While not directly therapeutic, PAP7 antibody studies have clarified mechanisms of hormone-induced steroidogenesis and mitochondrial dysfunction. For example, disruptions in PAP7-PKA-PBR interactions could underlie pathologies like adrenal insufficiency or neurodegenerative disorders .

Future Directions

Structural studies: Mapping PAP7’s PBR- and PKA-binding domains using epitope-specific antibodies.
Disease models: Investigating PAP7 expression in conditions like primary biliary cholangitis or iron overload syndromes .

Product Specs

Buffer

**Preservative:** 0.03% Proclin 300
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4

Form

Liquid

Lead Time

Made-to-order (14-16 weeks)

Synonyms

PAP7 antibody; At2g01880 antibody; T23K3.7 antibody; Purple acid phosphatase 7 antibody; EC 3.1.3.2 antibody

Target Names

PAP7

Uniprot No.

Q8S341

Target Background

Database Links

KEGG: ath:AT2G01880

STRING: 3702.AT2G01880.1

UniGene: At.42460

Protein Families

Metallophosphoesterase superfamily, Purple acid phosphatase family

Subcellular Location

Secreted.

Tissue Specificity

Expressed in roots, stems, leaves, flowers and siliques.

Q&A

Basic Research Questions

How do I validate PAP7 antibody specificity in Western blotting?

Method: Combine immunoblotting with epitope mapping using truncated protein variants or cyanogen bromide cleavage fragments .
Data: In yeast PAP studies, N-term antibodies recognized residues 1-100, while C-term antibodies bound the last 20 residues .
Controls: Include knockout cell lines or tissues lacking the target protein to confirm absence of cross-reactivity .

What experimental models are optimal for studying PAP7 antibody interactions?

In vitro: Use purified PAP7 protein in poly(A) polymerase activity assays (e.g., ATP-dependent RNA elongation) .
In vivo: Immunodepletion in yeast extracts reduces cleavage activity by 70%, restored by adding cleavage factor I (CF I) .
Cross-species reactivity: PAP7 antibodies show specificity for yeast and do not recognize mammalian or Xenopus homologs .

How are PAP7 antibody titers quantified in serum samples?

Protocol:
- Centrifuge blood at 3,000 rpm for 15 minutes at 4°C .
- Use ELISA with a threshold optical density (OD) of 0.45–0.60 at 450 nm .
- Validate via ROC curves (AUC ≥0.88 for combined autoantibodies) .

Advanced Research Questions

How to resolve discrepancies in PAP7 antibody performance across assays?

Case study: C-term antibodies deplete PAP7 activity in yeast extracts but fail to inhibit poly(A) addition in vitro .
Resolution:
- Test antibody-antigen complexes in the presence of cofactors (e.g., ATP, CF I) .
- Use thermal shift assays (ΔTm ≥5°C indicates stability issues) .

What computational tools optimize PAP7 antibody stability for longitudinal studies?

Approach:
- Apply the Sparrow Search Algorithm (SSA) or XGBoost to predict aggregation-prone regions .
- Engineer framework residues via covariance analysis (e.g., Abacus platform) to improve thermal stability (e.g., ΔDSF ≥10°C) .
Outcome: Engineered variants (e.g., MS-1797) show 2.5× higher production titers and reduced aggregation .

How to design a machine learning model for PAP7 antibody diagnostic applications?

Workflow:
- Train on serum autoantibody levels (e.g., MAGEA1, P53, PGP9.5) .
- Use SSA-XGBOOST for feature selection (AUC=0.9265 in lung adenocarcinoma) .
Validation: Compare with traditional ROC analysis (sensitivity: 84.05%, specificity: 91.85%) .

Key Data Tables

Table 1. PAP7 Antibody Performance in Functional Assays

Assay Type	N-term Antibody Activity	C-term Antibody Activity
Poly(A) addition	No inhibition	No inhibition
Cleavage in CF I	Reduced by 70%	Reduced by 70%

Table 2. Machine Learning Features for PAP7 Diagnostic Models

Autoantibody	Feature Weight (%)	AUC Contribution
MAGEA1	32.1	0.8870
P53	28.6	0.8265
PGP9.5	19.3	0.7740

Methodological Recommendations

For epitope mapping, use truncated PAP7 variants and mass spectrometry .
In diagnostic studies, prioritize combined autoantibody panels over single markers .
For therapeutic development, adopt Fc-Xtend mutations to extend serum half-life .

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PAP7 Antibody