ANP32D Antibody

What is ANP32D and how does it differ from other ANP32 family members?

ANP32D (acidic leucine-rich nuclear phosphoprotein 32 family member D) is a 14 kDa protein belonging to the acidic nuclear phosphoprotein 32 family. Unlike its family member PP32 (ANP32A), which functions as a tumor suppressor, ANP32D demonstrates tumorigenic properties. The critical distinction lies in the absence of a specific 25 amino acid region in ANP32D that is present in PP32 and responsible for its tumor suppression function . ANP32D is also known as PP32R2 (Phosphoprotein 32-related protein 2) or tumorigenic protein pp32r2.

While ANP32A and ANP32B are essential for influenza virus replication and interact with viral polymerase complexes, current research does not indicate ANP32D plays a similar role in viral replication . ANP32D's functional divergence from other family members makes it a distinct target for cancer research applications.

What applications are ANP32D antibodies validated for?

ANP32D antibodies such as the PACO47302 are validated for multiple research applications:

These applications enable researchers to investigate ANP32D expression patterns, subcellular localization, and protein interactions in various experimental contexts . When designing experiments, researchers should consider optimizing the antibody dilution within the recommended range based on their specific sample type and detection method.

How should ANP32D antibodies be stored and handled to maintain activity?

For optimal antibody performance and longevity, ANP32D antibodies should be stored in their recommended buffer conditions. For example, the PACO47302 antibody is stored in a preservative solution containing 0.03% Proclin 300, 50% glycerol, and 0.01M PBS at pH 7.4 .

Methodological approach to antibody storage:

• Store antibody aliquots at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles (create single-use aliquots)

• For short-term use (1-2 weeks), store at 4°C

• Protect from light, particularly fluorophore-conjugated variants

• Centrifuge briefly before opening to collect solution at the bottom of the tube

• Avoid introducing contaminants into the stock solution

When diluting the antibody, use freshly prepared buffer solutions, preferably containing a carrier protein (like BSA) to prevent nonspecific adsorption to container surfaces.

What tissue types and samples express ANP32D?

ANP32D expression has been documented in several human tissues and cancer samples. Immunohistochemical analysis with ANP32D antibodies has successfully detected the protein in:

• Human gastric cancer tissue sections

• Human cell lines, particularly HeLa cells

• Various cancer cell models

Unlike ANP32A and ANP32B that are widely expressed across multiple tissue types, ANP32D demonstrates a more restricted expression pattern. This makes it a potentially specific marker for certain cancer types. When investigating a new tissue or cell type, researchers should include appropriate positive and negative controls to validate ANP32D expression.

How can ANP32D antibodies be used to investigate its tumorigenic properties compared to the tumor suppressor functions of ANP32A?

To investigate the contrasting functions of ANP32D (tumorigenic) versus ANP32A (tumor suppressor), researchers can employ a multi-faceted approach using ANP32D antibodies:

• Differential expression analysis: Use ANP32D and ANP32A antibodies in matched normal/tumor tissue arrays to quantify relative expression patterns. Correlate expression with tumor grade, stage, and patient outcomes.

• Co-immunoprecipitation studies: Employ ANP32D antibodies to pull down protein complexes and analyze interaction partners that differ from ANP32A complexes. This can reveal unique signaling pathways and molecular mechanisms.

• Dual immunofluorescence: Perform simultaneous labeling of ANP32D and ANP32A to examine potential mutual exclusivity or co-expression patterns at the single-cell level.

• Functional domain analysis: Utilize truncated ANP32D constructs with and without the 25 amino acid region absent in wild-type ANP32D (but present in ANP32A) to dissect functional differences. ANP32D antibodies can verify expression and localization of these constructs.

• Phosphorylation studies: Examine post-translational modifications that may distinguish ANP32D's tumorigenic activities using phospho-specific antibodies alongside total ANP32D antibodies.

The critical methodological consideration is to include proper controls that account for potential antibody cross-reactivity between family members due to their sequence homology .

What are the optimal protocols for using ANP32D antibodies in cancer research applications?

For cancer research applications, ANP32D antibodies can be employed with the following optimized protocols:

Immunohistochemistry Protocol for Formalin-Fixed Paraffin-Embedded (FFPE) Tissues:

• Deparaffinize sections and perform antigen retrieval (citrate buffer pH 6.0, 95°C, 20 minutes)

• Block endogenous peroxidase (3% H₂O₂, 10 minutes)

• Protein block (5% normal goat serum, 1 hour)

• Incubate with ANP32D primary antibody (1:100 dilution, overnight at 4°C)

• Apply HRP-conjugated secondary antibody (1:500, 1 hour at room temperature)

• Develop with DAB and counterstain with hematoxylin

• Score expression in relation to tumor/normal boundaries and correlate with pathological features

Immunofluorescence Protocol for Cancer Cell Lines:

• Fix cells (4% paraformaldehyde, 15 minutes)

• Permeabilize (0.1% Triton X-100, 10 minutes)

• Block (3% BSA, 1 hour)

• Incubate with ANP32D antibody (1:100 dilution, overnight at 4°C)

• Apply fluorophore-conjugated secondary antibody (1:500, Alexa Fluor 488, 1 hour)

• Counterstain nuclei with DAPI

• Image on confocal microscope for precise subcellular localization

For gastric cancer and HeLa cells, these protocols have been validated to produce specific ANP32D labeling . The key methodological consideration is to include matched isotype controls and antigen pre-absorption controls to confirm staining specificity.

How can researchers differentiate between ANP32D and other ANP32 family members in experimental settings?

Distinguishing between highly homologous ANP32 family members requires careful methodological approaches:

• Antibody selection: Choose ANP32D antibodies raised against unique epitopes not conserved in ANP32A/B/E. Antibodies targeting the C-terminal region are optimal since this region shows greatest sequence divergence among family members.

• Western blot differentiation: ANP32D has a molecular weight of approximately 14 kDa, distinguishing it from ANP32A/B (~30 kDa). Use gradient gels (4-20%) for optimal separation.

• qRT-PCR validation: Design primers targeting unique regions of ANP32D mRNA to confirm antibody specificity at the transcript level. This cross-validation approach ensures the detected protein is genuinely ANP32D.

• Knockout/knockdown controls: Generate CRISPR/siRNA models with selective depletion of individual ANP32 family members to confirm antibody specificity.

• Mass spectrometry confirmation: For critical experiments, immunoprecipitate using ANP32D antibodies and confirm protein identity by mass spectrometry peptide analysis.

The sequence homology between ANP32 proteins necessitates rigorous validation, particularly when examining tissues that express multiple family members simultaneously .

What are the potential pitfalls in ANP32D antibody-based experiments and how can they be mitigated?

Several methodological challenges exist when working with ANP32D antibodies:

• Cross-reactivity with other ANP32 family members: Due to sequence homology, antibodies may recognize multiple family proteins. Mitigation strategy: Validate antibody specificity using recombinant proteins of all ANP32 family members in parallel Western blots.

• Nonspecific binding in high-background tissues: Some tissues (e.g., liver) naturally exhibit high background. Mitigation strategy: Optimize blocking conditions using tissue-specific blockers and extend blocking time to 2 hours.

• Epitope masking due to protein-protein interactions: ANP32D's interactions may obscure antibody binding sites. Mitigation strategy: Test multiple antibodies targeting different epitopes.

• Fixation-dependent epitope changes: Some epitopes may be sensitive to fixation methods. Mitigation strategy: Compare different fixation protocols (paraformaldehyde, methanol, acetone) to determine optimal epitope preservation.

• Low signal strength due to low expression levels: ANP32D may be expressed at low levels in some tissues. Mitigation strategy: Implement signal amplification methods like tyramide signal amplification or polymer-based detection systems.

• Batch-to-batch variability in polyclonal antibodies: Polyclonal antibodies like PACO47302 may show batch variations. Mitigation strategy: Validate each new lot against a reference sample and maintain consistent lot usage throughout a study.

A systematic validation approach using multiple detection methods will ensure robust and reproducible results when working with ANP32D antibodies .

How can ANP32D antibodies be used to investigate potential roles in transcriptional regulation and apoptosis?

ANP32D is involved in transcriptional regulation and apoptotic pathways. Researchers can use ANP32D antibodies to investigate these functions through several methodological approaches:

• Chromatin Immunoprecipitation (ChIP):

  • Use ANP32D antibodies to precipitate chromatin fragments

  • Sequence associated DNA (ChIP-seq) to identify genomic binding sites

  • Analyze binding motifs to determine transcriptional networks regulated by ANP32D

  • Compare with ANP32A/B binding patterns to identify unique targets

• Proximity Ligation Assay (PLA):

  • Detect in situ interactions between ANP32D and transcription factors or apoptotic regulators

  • Quantify interaction frequency under different cellular stresses

  • Compare interaction patterns in normal versus cancer cells

• Subcellular Fractionation and Western Blot:

  • Separate nuclear, cytoplasmic, and chromatin-bound fractions

  • Probe with ANP32D antibodies to track redistribution during apoptosis

  • Monitor post-translational modifications that regulate ANP32D function

• TUNEL Assay with ANP32D Co-staining:

  • Perform TUNEL staining to identify apoptotic cells

  • Co-stain with ANP32D antibodies to correlate expression with apoptotic status

  • Quantify relative expression in apoptotic versus non-apoptotic cells

• Live-Cell Imaging with Tagged ANP32D and Antibody Validation:

  • Express fluorescently-tagged ANP32D in living cells

  • Induce apoptosis and track protein redistribution

  • Validate observations with fixed-cell antibody staining

Unlike ANP32A and ANP32B that have established roles in influenza virus replication , ANP32D's functions appear more centered on cellular growth control and apoptosis regulation, making these approaches particularly relevant for cancer biology investigations.

How do ANP32D research findings relate to potential therapeutic applications in cancer?

ANP32D's tumorigenic properties contrast with the tumor suppressor functions of other family members, positioning it as a potential therapeutic target. Methodological approaches using ANP32D antibodies can facilitate translational research:

• Biomarker development:

  • Quantify ANP32D expression in tumor tissue microarrays using standardized IHC protocols

  • Correlate expression with clinical outcomes to establish prognostic value

  • Develop scoring systems for potential clinical implementation

• Drug discovery screening:

  • Use ANP32D antibodies in high-content screening assays to identify compounds that modulate its expression or localization

  • Develop ELISA-based assays to quantify ANP32D-target protein interactions for inhibitor screening

  • Validate hits using functional assays in cancer cell models

• Therapeutic antibody development:

  • Perform epitope mapping to identify functionally critical regions of ANP32D

  • Generate and test neutralizing antibodies that inhibit ANP32D's tumorigenic functions

  • Evaluate antibody-drug conjugates targeting ANP32D-expressing cancer cells

The absence of the 25 amino acid tumor suppressor region in ANP32D provides a specific target for therapeutic development. Researchers should focus on this unique structural feature when designing interventions.

What are the emerging techniques for studying ANP32D protein-protein interactions?

Advanced methodologies for investigating ANP32D's interactome include:

• BioID proximity labeling:

  • Generate ANP32D-BioID fusion proteins

  • Identify proximal proteins through biotinylation

  • Confirm interactions with co-immunoprecipitation using ANP32D antibodies

  • Compare interactomes between normal and cancer cellular contexts

• CRISPR-based screening:

  • Perform genome-wide CRISPR screens in ANP32D-dependent cancer models

  • Identify synthetic lethal interactions

  • Validate hits using ANP32D antibodies to assess expression and localization changes

• Quantitative interactomics:

  • Use SILAC or TMT labeling with ANP32D immunoprecipitation

  • Quantify differential interactions under various cellular conditions

  • Create dynamic interaction networks that change during cancer progression

• Single-molecule imaging:

  • Employ super-resolution microscopy with ANP32D antibodies

  • Track individual ANP32D molecules and their interaction partners

  • Quantify interaction kinetics and spatial organization

• Cryo-EM structural analysis:

  • Similar to studies done with ANP32A in influenza replication complexes

  • Determine structural basis for ANP32D's unique functions

  • Identify structural differences from other family members that explain functional divergence

These approaches can reveal how ANP32D's interactions differ from those of ANP32A and ANP32B, which have established roles in processes like influenza virus replication .