DPPA3 Antibody, HRP conjugated

What is DPPA3 and what cellular functions does it perform?

DPPA3 (Developmental Pluripotency-Associated 3), also known as Stella, is an intrinsically disordered protein that plays a critical role in epigenetic regulation. It specifically interacts with UHRF1 (Ubiquitin-like with PHD and RING finger domain-containing protein 1) and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. The structural studies demonstrate that DPPA3 forms induced α-helices upon binding to the UHRF1 PHD domain, creating a multifaceted interaction interface unlike canonical PHD domain ligands . This unique binding mechanism allows DPPA3 to compete with histone H3 for UHRF1 binding, thereby regulating DNA methylation maintenance during cell proliferation.

What is the localization pattern of DPPA3 in mouse tissues?

Immunohistochemistry studies using specific antibodies have shown that DPPA3/Stella is primarily localized to oocytes in mouse ovary tissue. The protein can be detected using appropriate antibodies such as Goat Anti-Mouse Stella/DPPA3 Antigen Affinity-purified Polyclonal Antibody at 1 μg/mL concentration with overnight incubation at 4°C . The specific staining pattern is evident after heat-induced epitope retrieval and visualization with an HRP polymer detection system, showing clear localization to oocytes with minimal background in surrounding tissues.

How does the molecular structure of DPPA3 facilitate its biological function?

DPPA3's intrinsically disordered nature allows it to adopt specific conformations upon binding to target proteins. Structural analyses reveal that when DPPA3 interacts with the UHRF1 PHD domain, it forms two induced α-helices (αS1 and αL2) and utilizes a conserved 88VRT90 cassette for recognition by a shallow acidic groove on the UHRF1 PHD domain . The estimated contact area between DPPA3 and the UHRF1 PHD domain is approximately 1360 Å2, which is significantly larger than the contact areas for histone H3 (~400 Å2) and PAF15 (~360 Å2) . This extensive interaction surface contributes to DPPA3's high binding affinity (KD = 45 nM) and its ability to effectively compete with other ligands for UHRF1 binding.

What criteria should be used when selecting a DPPA3 antibody for specific applications?

When selecting a DPPA3 antibody for research applications, consider several critical factors to ensure experimental success. First, verify species reactivity—the antibody should recognize DPPA3 from your experimental organism (e.g., mouse DPPA3). Second, confirm application compatibility through validation data for your specific technique (IHC, Western blot, etc.). Third, examine the immunogen sequence to ensure the antibody targets relevant epitopes—for studying DPPA3-UHRF1 interactions, antibodies recognizing the C-terminal region (residues 76-128) would be most informative based on structural studies . For HRP-conjugated antibodies specifically, verify conjugation quality and enzyme activity retention. Finally, assess available validation data showing specificity in tissues known to express DPPA3, such as mouse ovary tissue where DPPA3 localizes to oocytes .

What validation experiments should be performed to confirm DPPA3 antibody specificity?

A comprehensive validation strategy for DPPA3 antibodies involves multiple complementary approaches. Begin with Western blot analysis to confirm detection of a single band at the expected molecular weight (~20 kDa for mouse DPPA3). Perform immunohistochemistry on positive control tissues (e.g., mouse ovary) where DPPA3 expression has been established, comparing staining patterns with published literature . Include appropriate negative controls such as isotype controls and tissues where DPPA3 is not expressed. For definitive validation, use genetic approaches such as DPPA3 knockout/knockdown samples to demonstrate loss of signal. When testing HRP-conjugated antibodies, include peroxidase inhibition steps to distinguish between specific signal and potential endogenous peroxidase activity. Finally, peptide competition assays can confirm epitope specificity by pre-incubating the antibody with the immunizing peptide.

How can researchers differentiate between specific and non-specific binding when using DPPA3 antibodies?

Distinguishing specific from non-specific binding requires systematic control experiments and optimization. First, include multiple negative controls: isotype-matched control antibodies, secondary-only controls for indirect detection methods, and biological negative controls (tissues known not to express DPPA3). Second, perform a titration series (0.1-10 μg/mL) to identify the optimal antibody concentration that maximizes specific signal while minimizing background. Third, optimize blocking conditions using different blocking agents (BSA, normal serum, commercial blockers) and durations. Fourth, when using HRP-conjugated antibodies, ensure complete quenching of endogenous peroxidase activity in tissues. Fifth, compare the observed staining pattern with the known biological distribution of DPPA3 (e.g., oocyte-specific localization in ovary tissue) . Finally, confirm specificity through orthogonal methods such as RNA expression data or multiple antibodies targeting different epitopes.

What is the optimal protocol for immunohistochemistry using HRP-conjugated DPPA3 antibody?

Based on published methodologies, an optimized IHC protocol for DPPA3 detection includes the following steps:

• Tissue Preparation: Use perfusion-fixed frozen sections of tissue (such as mouse ovary) for optimal epitope preservation.

• Antigen Retrieval: Perform heat-induced epitope retrieval using an appropriate buffer such as Antigen Retrieval Reagent-Basic before antibody incubation .

• Blocking: Block endogenous peroxidase activity with 0.3% H₂O₂ in methanol for 30 minutes, followed by protein blocking with 5-10% normal serum from the same species as the secondary antibody.

• Primary Antibody Incubation: For direct HRP-conjugated DPPA3 antibodies, apply at 1-5 μg/mL and incubate overnight at 4°C. For indirect detection, use unconjugated primary antibody at 1 μg/mL overnight at 4°C .

• Detection: For indirect methods, apply HRP-polymer detection reagent (e.g., Anti-Goat IgG HRP Polymer Antibody) according to manufacturer's instructions .

• Visualization: Develop with DAB (3,3'-diaminobenzidine) substrate for 5-10 minutes while monitoring for optimal signal development.

• Counterstaining: Counterstain with hematoxylin for 1-2 minutes to visualize tissue architecture .

• Mounting: Dehydrate through ascending alcohol series, clear in xylene, and mount with permanent mounting medium.

The expected result is specific staining localized to oocytes in ovary tissue with minimal background in surrounding tissue compartments.

How should antibody dilution be optimized for Western blot analysis of DPPA3?

Optimizing antibody dilution for Western blot analysis of DPPA3 requires a systematic approach:

• Sample Preparation: Extract proteins using RIPA buffer supplemented with protease inhibitors. For DPPA3 detection, include phosphatase inhibitors as phosphorylation may affect antibody recognition.

• Protein Loading: Load 20-50 μg of total protein per lane, with positive controls (tissue known to express DPPA3) and negative controls (DPPA3-knockout samples or tissues not expressing the protein).

• Dilution Series: Prepare a dilution series of HRP-conjugated DPPA3 antibody ranging from 1:500 to 1:5000 in blocking buffer (5% non-fat dry milk or BSA in TBST).

• Incubation: Apply different antibody dilutions to identical membrane strips containing the same samples, incubating overnight at 4°C with gentle agitation.

• Washing: Perform stringent washing (4-5 times for 5 minutes each) with TBST to remove unbound antibody.

• Detection: Develop using enhanced chemiluminescence substrate, controlling exposure times to prevent overexposure.

• Analysis: Compare signal-to-noise ratio across dilutions, selecting the dilution that provides clear detection of the expected ~20 kDa DPPA3 band with minimal background.

• Validation: Confirm specificity by peptide competition or using DPPA3 variants with mutations in key residues such as R89A/T90A or R104A, which affect protein function .

What controls are essential when using DPPA3 antibodies for chromatin immunoprecipitation (ChIP)?

When performing ChIP with DPPA3 antibodies, the following controls are essential:

• Input Control: Reserve 5-10% of chromatin before immunoprecipitation to normalize enrichment calculations.

• Positive Control Antibody: Include a ChIP-validated antibody against a well-characterized protein (e.g., histone H3) to confirm the protocol is working.

• Negative Control Antibody: Use isotype-matched IgG from the same species as the DPPA3 antibody to establish background enrichment levels.

• Positive Control Loci: Design primers for genomic regions where DPPA3 is known or predicted to bind, particularly regions containing UHRF1 binding sites, since DPPA3 functionally interacts with UHRF1 .

• Negative Control Loci: Include primers for genomic regions not expected to be bound by DPPA3 (e.g., housekeeping gene promoters or gene deserts).

• Biological Controls: When possible, include samples with modulated DPPA3 expression (overexpression or knockdown) to confirm enrichment specificity.

• Technical Replicates: Perform at least three technical replicates for each ChIP experiment to ensure reproducibility.

• Sequential ChIP: Consider performing sequential ChIP (Re-ChIP) for DPPA3 followed by UHRF1 to identify genomic regions where both proteins co-localize, providing insight into the functional interaction mechanisms demonstrated in structural studies .

What strategies can resolve weak or absent signal when using DPPA3 antibodies?

Resolving weak or absent DPPA3 signal requires a systematic troubleshooting approach addressing multiple aspects of the experimental procedure:

• Antibody Quality: Verify antibody integrity by checking for degradation (especially for HRP conjugates which can lose enzymatic activity). Test a new antibody lot or alternative clone if necessary.

• Epitope Accessibility: Optimize antigen retrieval methods, testing different buffers (citrate pH 6.0, EDTA pH 8.0, Tris pH 9.0) and retrieval conditions (microwave, pressure cooker, water bath). This is particularly important for DPPA3 detection since structural studies show that DPPA3 undergoes conformational changes upon binding to interaction partners .

• Antibody Concentration: Increase antibody concentration incrementally (e.g., from 1 μg/mL to 5 μg/mL) while monitoring background levels .

• Detection System: For HRP-conjugated antibodies, ensure substrate freshness and consider switching to more sensitive detection systems such as tyramide signal amplification.

• Incubation Conditions: Extend primary antibody incubation time (overnight at 4°C to 48 hours) or try room temperature incubation to promote binding kinetics.

• Sample Preparation: Verify protein integrity by testing different extraction and fixation methods to preserve DPPA3 epitopes.

• Expression Levels: Confirm DPPA3 expression in your sample using RT-qPCR or other methods, as expression may be developmentally regulated or cell-type specific.

• Blocking Conditions: Test alternative blocking reagents (BSA, casein, commercial blockers) to prevent non-specific interactions without interfering with antibody binding.

How can researchers minimize background when using HRP-conjugated DPPA3 antibodies?

Minimizing background with HRP-conjugated DPPA3 antibodies requires attention to several key factors:

• Endogenous Peroxidase Quenching: Thoroughly block endogenous peroxidase activity using 0.3-3% H₂O₂ in methanol for 30 minutes before antibody application, especially in tissues with high peroxidase content.

• Optimal Antibody Concentration: Perform careful titration experiments to identify the minimum effective concentration that provides specific signal while minimizing background. For DPPA3 detection, starting with 1 μg/mL has been effective in published protocols .

• Blocking Optimization: Test different blocking agents (5-10% normal serum, 1-5% BSA, commercial blockers) and extend blocking time to 1-2 hours at room temperature.

• Buffer Additives: Include 0.1-0.3% detergent (Triton X-100 or Tween-20) in washing and dilution buffers to reduce non-specific hydrophobic interactions.

• Wash Stringency: Increase wash duration and number of wash steps (5 washes of 5 minutes each) with gentle agitation to remove unbound antibody.

• Antibody Diluent: Use commercial antibody diluents designed to minimize background or add 0.1-1% BSA to washing buffers.

• Substrate Development: Carefully control substrate incubation time, stopping the reaction before background develops while maintaining specific signal.

• Tissue Preparation: Ensure complete deparaffinization and hydration of tissue sections, as incomplete processing can lead to non-specific binding and high background.

How can researchers distinguish between genuine DPPA3 signal and artifacts in immunofluorescence microscopy?

Distinguishing genuine DPPA3 signal from artifacts in immunofluorescence requires rigorous controls and careful analysis:

• Biological Pattern Verification: Confirm that the observed DPPA3 localization matches expected patterns based on published data, such as oocyte-specific localization in ovary tissue .

• Z-stack Analysis: Perform z-stack imaging to distinguish between genuine signal throughout the cell and surface artifacts that appear only in specific focal planes.

• Multiple Channel Controls: Include single-channel controls when performing multi-color immunofluorescence to identify potential bleed-through between fluorophores.

• Autofluorescence Assessment: Examine unstained sections to identify sources of tissue autofluorescence, particularly in fixatives containing aldehydes.

• Peptide Competition: Pre-incubate antibody with immunizing peptide to confirm signal specificity—genuine DPPA3 signal should be eliminated or significantly reduced.

• Genetic Controls: When possible, include DPPA3 knockout or knockdown samples as definitive negative controls.

• Alternative Antibodies: Verify results using antibodies targeting different DPPA3 epitopes—genuine signal should show consistent patterns across antibodies.

• Correlation with Functional Data: Correlate observed DPPA3 localization with functional data, such as the presence of UHRF1 and DNA methylation patterns, based on the known role of DPPA3 in regulating UHRF1 chromatin localization .

How can DPPA3 antibodies be used to study its role in DNA methylation regulation?

DPPA3 antibodies provide valuable tools for investigating its regulatory role in DNA methylation through several advanced approaches:

• ChIP-seq Analysis: Use DPPA3 antibodies for chromatin immunoprecipitation followed by sequencing to map genome-wide binding sites. Compare these with UHRF1 binding patterns and DNA methylation profiles to identify regions where DPPA3 actively regulates methylation. Based on structural studies, DPPA3 competes with histone H3 for binding to UHRF1's PHD domain, providing a mechanism for its inhibitory effect on DNA methylation .

• Co-Immunoprecipitation: Employ DPPA3 antibodies for co-IP experiments to isolate protein complexes containing DPPA3 and analyze interacting partners beyond UHRF1, potentially revealing additional regulatory mechanisms.

• Immunofluorescence Co-localization: Perform dual immunofluorescence with DPPA3 and UHRF1 antibodies to analyze their spatial relationship during development or in response to cellular stressors. The structural data suggesting competition between DPPA3 and histone H3 for UHRF1 binding can be functionally validated through these co-localization studies .

• Proximity Ligation Assay: Use DPPA3 antibodies in proximity ligation assays to visualize and quantify direct protein-protein interactions with UHRF1 in situ, providing spatial information about where in the cell these interactions occur.

• FLIM-FRET Analysis: Combine DPPA3 antibodies with fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET) to measure the distance between DPPA3 and UHRF1 in living cells, providing dynamic information about their interaction.

• Mass Spectrometry: Use DPPA3 antibodies for immunoprecipitation followed by mass spectrometry to identify post-translational modifications on DPPA3 that might regulate its interaction with UHRF1 and effect on DNA methylation.

• Chromatin Fractionation: Combine subcellular fractionation with DPPA3 immunoblotting to analyze how DPPA3 affects UHRF1 distribution between chromatin-bound and soluble fractions, directly testing the model that DPPA3 inhibits UHRF1 chromatin localization .

What approaches can be used to study the structural interactions between DPPA3 and UHRF1 using antibodies?

Studying the structural interactions between DPPA3 and UHRF1 requires sophisticated approaches that leverage antibodies as analytical tools:

How can DPPA3 antibodies contribute to understanding its role in cancer biology?

DPPA3 antibodies offer valuable tools for investigating its emerging role in cancer biology through several approaches:

• Tissue Microarray Analysis: Use DPPA3 antibodies for immunohistochemical analysis of cancer tissue microarrays to assess expression patterns across multiple cancer types and correlate with clinical outcomes. This is particularly relevant given the finding that DPPA3 overexpression leads to tumor differentiation in hepatocellular carcinoma by impeding UHRF1 nuclear translocation .

• Methylation Status Correlation: Combine DPPA3 immunostaining with DNA methylation analysis to investigate how DPPA3 expression correlates with global and gene-specific DNA methylation patterns in cancer tissues, given its role in regulating UHRF1-dependent DNA methylation .

• Therapeutic Response Biomarker: Evaluate DPPA3 expression as a potential biomarker for response to epigenetic therapies such as DNA methyltransferase inhibitors, based on its role in regulating DNA methylation.

• Functional Imaging: Develop fluorescently labeled anti-DPPA3 antibodies for in vivo imaging to track DPPA3 expression and localization during tumor progression in animal models.

• Circulating Tumor Cell Analysis: Investigate whether DPPA3 can be detected in circulating tumor cells using specific antibodies, potentially providing a liquid biopsy approach for cancers where DPPA3 plays a functional role.

• Drug Development Platform: Based on structural insights showing how DPPA3 inhibits UHRF1, use antibodies to screen for compounds that mimic DPPA3's interaction with UHRF1 as potential anticancer drugs . The structural data showing that αL2 of DPPA3 bridges the pre- and core-PHD domains of UHRF1 provides a rational basis for designing peptide-like inhibitors.

• Combination Therapy Assessment: Use antibodies to monitor how modulating DPPA3 function affects response to conventional cancer therapies, potentially identifying synergistic treatment approaches.

Binding parameters of DPPA3 with UHRF1 compared to other PHD domain ligands

Effect of mutations on DPPA3-UHRF1 interaction

Optimization parameters for DPPA3 antibody applications