PDAP1 Antibody

What is PDAP1 and what are its key biological functions?

PDAP1 (PDGFA Associated Protein 1) is a highly conserved phosphoprotein across vertebrate species with multiple important biological functions. It was originally characterized as a casein kinase II substrate in the rat brain and was shown to bind PDGFA with low affinity in a rat neural retina cell line .

The protein has several key functions:

• Enhances PDGFA-stimulated cell growth in fibroblasts while inhibiting the mitogenic effect of PDGFB

• Acts as an essential regulator of cellular homeostasis in mature B cells

• Protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification

• Plays a crucial role in hepatitis A virus (HAV) infection

• Functions in tumor initiation and progression in colorectal cancer (CRC)

PDAP1 is a 21 kDa protein (calculated molecular weight) that typically appears at 25-28 kDa in Western blots due to post-translational modifications . It is also known by several aliases including 28 kDa heat- and acid-stable phosphoprotein, PAP, PAP1, and HASPP28 .

What are the typical applications for PDAP1 antibodies in research?

PDAP1 antibodies are versatile research tools that can be used across multiple applications:

Methodological considerations: When selecting an application, researchers should consider tissue-specific expression levels. For low-expressing tissues, more sensitive techniques like immunoprecipitation followed by Western blotting may be preferable to direct Western blotting .

How should PDAP1 antibody specificity be validated?

Proper validation is essential for ensuring reliable results with PDAP1 antibodies:

Recommended validation methods:

• Knockout/knockdown controls: Use PDAP1 knockout cell lines (e.g., PDAP1-KO1.4 and PDAP1-KO2 as described in hepatitis A virus studies) to confirm antibody specificity

• Peptide competition: Pre-incubate antibody with immunizing peptide to demonstrate specificity

• Cross-species reactivity testing: Verify specificity across human, mouse, and rat samples when using antibodies claiming multi-species reactivity

• Multiple antibody concordance: Use different antibodies targeting different PDAP1 epitopes to confirm findings

Key validation metrics:

• Observe expected molecular weight (calculated: 21 kDa; observed: typically 25-28 kDa)

• Verify subcellular localization pattern

• Confirm absence of signal in knockout/knockdown samples

• Check for consistency of results across multiple applications

How can PDAP1 antibodies be used to investigate its role in B cell function and antibody diversification?

PDAP1 has been identified as an essential regulator of cellular homeostasis in mature B cells. Research shows that Pdap1 deficiency leads to sustained expression of the integrated stress response (ISR) effector activating transcription factor 4 (Atf4), resulting in increased cell death and defective AID expression .

Methodological approach:

• Isolation of B cells: Purify B cells from wild-type and Pdap1-conditional knockout mice

• Stress induction: Treat cells with known ISR activators (e.g., thapsigargin, tunicamycin)

• Analysis of ISR pathway: Use PDAP1 antibodies in combination with antibodies against:

  • Phosphorylated eIF2α

  • ATF4

  • CHOP

  • Activation-induced cytidine deaminase (AID)

• B cell functional assays:

  • Class switch recombination (CSR) analysis

  • Somatic hypermutation (SHM) assessment

  • Germinal center B cell formation

Data interpretation considerations:

• Compare phosphorylation status of ISR components between wild-type and Pdap1-deficient B cells

• Assess correlation between PDAP1 expression levels and B cell survival under stress

• Evaluate impact on antibody diversification mechanisms through AID expression

What methods can be employed to study PDAP1's role in cancer progression using specific antibodies?

Recommended methodological workflow:

• Expression analysis:

  • Immunohistochemistry of tumor tissue microarrays using PDAP1 antibodies

  • Quantitative Western blotting to compare expression levels between normal and cancer tissues

  • Correlation with patient clinical data including survival analysis

• Mechanistic studies:

  • Co-immunoprecipitation with PDAP1 antibodies to identify interacting partners

  • Proximity ligation assay to confirm PDAP1-EGFR interaction in situ

  • Phosphoprotein profiling following PDAP1 knockdown/overexpression

• Functional validation:

  • Cell proliferation, migration, and invasion assays in cells with modulated PDAP1 expression

  • Xenograft models with PDAP1 knockdown cancer cells

  • Patient-derived xenograft (PDX) models treated with PDAP1-targeting agents

Research findings from CRC studies:

How can researchers use PDAP1 antibodies to investigate its role in viral infection mechanisms?

Recent research has revealed PDAP1 as a critical host factor for hepatitis A virus (HAV) infection, with near-absolute requirement for PDAP1 in HAV infection and pathogenesis .

Methodological approach for viral studies:

• Infection models:

  • Cell culture: Compare HAV replication in wild-type vs. PDAP1-knockout cell lines

  • Animal models: Use conditional knockout mice (e.g., Alb-Cre+Pdap1 mice) for in vivo studies

• Viral replication assessment:

  • Viral RNA quantification by qRT-PCR

  • Infectious virus production using reporter viruses (e.g., 18f-NLuc)

  • Viral protein detection using immunoblotting

• Mechanistic investigations:

  • Co-immunoprecipitation of PDAP1 with viral proteins using specific antibodies

  • Immunofluorescence co-localization studies of PDAP1 and viral components

  • Analysis of translation initiation complexes in the context of IRES-dependent translation

Key research findings:

• PDAP1-KO cells produced 50-75 fold less HAV RNA than control cells

• Alb-Cre+Pdap1 mice were completely refractory to infectious challenge with HAV

• HAV IRES-directed translation requires PDAP1, which may interact with translation initiation factors

• PDAP1 requirement appears specific to HAV, as poliovirus replication was not affected in PDAP1-KO cells

What considerations are important when using PDAP1 antibodies for investigating protein-protein interactions?

PDAP1 functions through interactions with multiple proteins, including EGFR in cancer progression and potentially translation factors in viral infection. Investigating these interactions requires careful experimental design.

Methodological considerations:

• Co-immunoprecipitation optimization:

  • Select antibodies targeting different epitopes of PDAP1 to avoid interference with protein binding sites

  • Consider native vs. cross-linking conditions depending on interaction stability

  • Use appropriate controls (IgG control, PDAP1-knockout cells)

• Proximity-based methods:

  • Proximity ligation assay (PLA) for in situ detection of protein interactions

  • FRET/BRET approaches for dynamic interaction studies

  • BioID or APEX2 proximity labeling to identify novel interaction partners

• Domain mapping strategies:

  • Use antibodies specific to different regions of PDAP1 (N-terminal, C-terminal) to understand which domains mediate specific interactions

  • Test interactions with truncated versions of PDAP1 and its partners

Research findings on PDAP1 interactions:

• PDAP1 interacts with the juxtamembrane domain of EGFR and facilitates EGFR-MAPK signaling activation in CRC cells

• PDAP1 binding to PDGFA occurs with low affinity in rodent models

• PDAP1 is phosphorylated in vitro by several kinases, including PKC, PKA, CKI, and CKII, with CKII being the major kinase in intact cells

What are common issues when using PDAP1 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with PDAP1 antibodies:

Methodological guidance:

• For immunohistochemistry, suggested antigen retrieval with TE buffer pH 9.0 often works best, though citrate buffer pH 6.0 may be used as an alternative

• For Western blotting, dilutions between 1:500-1:2000 are typically effective

• For immunofluorescence, dilutions of 1:200-1:800 typically provide optimal results

How should researchers interpret the discrepancy between calculated and observed molecular weights for PDAP1?

PDAP1 has a calculated molecular weight of 21 kDa, but is frequently observed at 25-28 kDa in Western blots . This discrepancy is important to understand when interpreting results.

Explaining the molecular weight difference:

• Post-translational modifications: PDAP1 is a phosphoprotein and may undergo multiple phosphorylation events by kinases including CKII, PKC, PKA, and CKI

• Structural properties: As a heat- and acid-stable protein, PDAP1 may have unusual structural characteristics affecting electrophoretic mobility

• Isoforms: Potential alternative splicing or processing variants

Methodological approaches to confirm specificity:

• Dephosphorylation treatment: Treat samples with phosphatases before Western blotting to see if apparent molecular weight shifts

• Knockout/knockdown validation: Confirm band disappearance in PDAP1-depleted samples

• Mass spectrometry: Confirm protein identity and modifications following immunoprecipitation

How can PDAP1 antibodies be effectively used in multiplexing experiments?

Investigating PDAP1's role in complex signaling pathways often requires simultaneous detection of multiple proteins.

Multiplexing methodological strategies:

• Co-immunofluorescence optimization:

  • Select primary antibodies from different host species (e.g., rabbit anti-PDAP1 with mouse anti-EGFR)

  • Use directly conjugated antibodies (e.g., CoraLite® Plus 488-conjugated PDAP1 antibody)

  • Implement sequential staining protocols for same-species antibodies

• Western blot multiplexing:

  • Strip and reprobe membranes (suitable for proteins of different molecular weights)

  • Use fluorescent secondary antibodies with different emission spectra

  • Consider antibodies against phosphorylated and total proteins for signaling studies

• Flow cytometry panels:

  • Carefully select fluorophore combinations to minimize spectral overlap

  • Include appropriate compensation controls

  • Consider cell permeabilization protocols optimized for PDAP1 detection

Research application example:
For studying PDAP1's role in EGFR-MAPK-FRA-1 signaling in CRC cells, researchers successfully used co-immunoprecipitation with PDAP1 antibodies followed by immunoblotting for EGFR, and phosphoprotein antibody arrays to identify downstream effectors in the signaling cascade .

How are PDAP1 antibodies being used to investigate novel therapeutic approaches?

Recent studies suggest PDAP1 as a potential therapeutic target, particularly in cancers where it is overexpressed.

Methodological approaches for therapeutic investigations:

• Target validation studies:

  • Use PDAP1 antibodies to correlate expression with patient outcomes in clinical samples

  • Employ PDAP1 knockdown/knockout followed by phenotypic characterization

  • Analyze PDAP1-dependent pathways in diverse cancer models

• Therapeutic monitoring:

  • Develop quantitative assays using PDAP1 antibodies to monitor target engagement

  • Track PDAP1 expression levels during treatment response

  • Investigate combination therapies targeting PDAP1-related pathways

• Patient selection biomarkers:

  • Immunohistochemical assessment of PDAP1 expression for patient stratification

  • Correlation of PDAP1 levels with response to targeted therapies

Research findings with therapeutic implications:

• Silencing of PDAP1 hindered the growth of patient-derived xenografts with high PDAP1 levels

• PDAP1 inhibition could be warranted for CRC patients with PDAP1 overexpression

• Conditional knockout of Pdap1 in intestinal epithelial cells impaired mucosal restitution and inhibited tumor growth in colitis-associated cancers

What methods can be used to study the evolutionary conservation of PDAP1 across species?

PDAP1 is highly conserved across vertebrate species, suggesting important fundamental biological functions.

Methodological approach for comparative studies:

• Antibody cross-reactivity assessment:

  • Test antibodies against recombinant PDAP1 from different species

  • Verify specificity in tissues/cells from various species (human, mouse, rat, zebrafish, etc.)

  • Map conserved epitopes recognized by broadly reactive antibodies

• Functional conservation studies:

  • Compare PDAP1 expression patterns across species using validated antibodies

  • Analyze post-translational modifications across species

  • Assess functional complementation by expressing PDAP1 from different species in knockout models

Available antibody resources for cross-species studies:

• Antibodies with verified reactivity to human, mouse, and rat PDAP1

• Antibodies with broader reactivity including cow, dog, guinea pig, horse, rabbit, zebrafish, bat, chicken, monkey, and pig

How can researchers investigate contradictory findings about PDAP1 function in different tissue contexts?

PDAP1 appears to have context-dependent functions across different tissues and disease states, which can lead to apparently contradictory findings.

Methodological approaches to resolve contradictions:

• Tissue-specific conditional knockout models:

  • Use tissue-specific Cre lines (e.g., Alb-Cre for liver, CD19-Cre for B cells)

  • Compare phenotypes across different conditional knockout models

  • Use PDAP1 antibodies to confirm tissue-specific deletion

• Context-dependent interaction studies:

  • Compare PDAP1 interactome across different cell types using immunoprecipitation and mass spectrometry

  • Identify tissue-specific post-translational modifications

  • Map different functional domains required in specific contexts

• Single-cell analysis approaches:

  • Use PDAP1 antibodies for single-cell protein analysis in heterogeneous tissues

  • Correlate with single-cell transcriptomics data

  • Identify cell type-specific functions and expression patterns

Research examples illustrating context-dependent functions:

• In B cells, PDAP1 protects against stress-induced cell death and promotes antibody diversification

• In liver cells, PDAP1 is essential for hepatitis A virus infection

• In colorectal cancer, PDAP1 promotes proliferation, migration, invasion, and metastasis

These distinct functions highlight the importance of tissue context when investigating PDAP1 biology and interpreting antibody-based studies.