dpysl3 Antibody

What is DPYSL3 and why is it important for research?

DPYSL3 (Dihydropyrimidinase-Like 3) is a cytoplasmic protein with a canonical length of 570 amino acids and a molecular mass of approximately 62 kDa in humans. It belongs to the Hydantoinase/dihydropyrimidinase protein family and plays crucial roles in cytokine-mediated signaling pathways and regulation of cell migration . Also known as CRMP4, DRP-3, ULIP, and several other synonyms, DPYSL3 is predominantly expressed in heart and skeletal muscle tissues. Up to two different isoforms have been reported for this protein, allowing for tissue-specific regulation and function . DPYSL3's involvement in cellular migration pathways makes it particularly relevant for developmental biology, neuroscience, and cancer research applications.

What applications are most suitable for DPYSL3 antibodies?

DPYSL3 antibodies are adaptable to multiple research applications with varying degrees of optimization requirements. Western Blot (WB) is the most widely used application, providing quantitative data on DPYSL3 expression levels and molecular weight confirmation . Immunohistochemistry (IHC) allows visualization of DPYSL3 distribution in tissue sections, which is particularly valuable for studying expression patterns in heart and skeletal muscle . Enzyme-Linked Immunosorbent Assay (ELISA) offers highly sensitive quantitative detection, while Immunocytochemistry (ICC) enables subcellular localization studies in cultured cells . When selecting an application, researchers should consider the specific epitope binding region of their antibody, as this may affect recognition in fixed versus denatured samples.

How should researchers choose between polyclonal and monoclonal DPYSL3 antibodies?

The selection between polyclonal and monoclonal DPYSL3 antibodies depends on the experimental goals and required specificity. Polyclonal antibodies, such as many rabbit-derived anti-DPYSL3 products, recognize multiple epitopes on the DPYSL3 protein, providing stronger signal amplification and greater tolerance to minor protein denaturation . These characteristics make polyclonals suitable for detecting low-abundance DPYSL3 in tissues where expression is minimal. Monoclonal antibodies, like the mouse monoclonal 1B8 against AA 457-555, offer superior specificity by recognizing a single epitope, reducing cross-reactivity with other CRMP family proteins . For experiments requiring absolute specificity (such as distinguishing between DPYSL3 and other CRMP family members), monoclonal antibodies that have been validated not to react with CRMP-1, CRMP-2, CRMP-3, or CRMP-5 would be optimal .

What species reactivity considerations are important when selecting DPYSL3 antibodies?

Species reactivity is a critical selection criterion for DPYSL3 antibodies, particularly in comparative or translational research. Available antibodies demonstrate varying reactivity profiles, from narrow species specificity to broad cross-reactivity . For human-focused research, multiple options exist with confirmed human DPYSL3 reactivity . Mouse and rat model researchers should select antibodies validated specifically for these species, as not all human-reactive antibodies cross-react with rodent orthologs . Some antibodies offer exceptionally broad reactivity across multiple species including human, mouse, rat, bovine, dog, guinea pig, horse, and rabbit, making them valuable for comparative studies . Always verify the documented reactivity pattern for your specific experimental model, as sequence variations between species can affect epitope recognition and antibody performance.

How do different epitope regions affect DPYSL3 antibody performance?

The epitope binding region significantly impacts DPYSL3 antibody performance across different experimental platforms. Antibodies targeting the N-terminal region (AA 1-218, AA 1-280) often provide robust detection in Western blotting but may be less effective in applications where protein folding preserves tertiary structure . C-terminal targeting antibodies (including those recognizing AA 457-555, AA 461-490, AA 465-570) typically demonstrate excellent performance in both denaturing and native conditions, making them versatile across multiple applications . The middle region antibodies may offer unique advantages for detecting specific functional domains involved in protein-protein interactions. When troubleshooting detection issues, consider switching to an antibody recognizing a different epitope, as post-translational modifications or protein interactions may mask certain regions in your experimental system.

What are recommended protocols for optimizing Western blots with DPYSL3 antibodies?

Optimizing Western blot protocols for DPYSL3 detection requires attention to several critical parameters. Begin with protein extraction using RIPA buffer supplemented with protease inhibitors to prevent degradation of the 62 kDa DPYSL3 protein . Load 20-40 μg of total protein per lane on 10-12% SDS-PAGE gels for optimal separation. During transfer, use PVDF membranes for their superior protein retention properties compared to nitrocellulose. For blocking, 5% non-fat dry milk in TBST is typically effective, though some DPYSL3 antibodies perform better with BSA-based blocking solutions . Primary antibody concentrations require careful titration, typically starting at 1:1000 for most commercial antibodies and adjusting based on signal-to-noise ratio. Extended primary antibody incubation overnight at 4°C often improves specific signal detection. For heart and skeletal muscle samples where DPYSL3 is highly expressed, shorter exposure times may be necessary to prevent signal saturation .

How can researchers validate DPYSL3 antibody specificity?

Validating DPYSL3 antibody specificity requires a multi-faceted approach to ensure reliable experimental results. Begin with positive and negative control samples - heart and skeletal muscle tissue lysates serve as excellent positive controls due to their high DPYSL3 expression . For negative controls, consider tissues with minimal DPYSL3 expression or DPYSL3 knockdown/knockout samples. Peptide competition assays, where the immunizing peptide blocks antibody binding, can confirm epitope specificity. When testing new antibodies, verify that they detect a band at the expected molecular weight of 62 kDa . Some antibodies specifically react with the 65 kDa CRMP-4 form from human, rat, and mouse samples without cross-reacting with other CRMP family members (CRMP-1, CRMP-2, CRMP-3, or CRMP-5) . Multiple antibody validation is also recommended - comparing results from antibodies targeting different DPYSL3 epitopes can provide confidence in specificity and increase result reliability.

What are the optimal fixation and permeabilization methods for DPYSL3 immunodetection?

The choice of fixation and permeabilization methods significantly impacts DPYSL3 immunodetection in IHC and ICC applications. For formalin-fixed paraffin-embedded (FFPE) tissues, antigen retrieval is essential, with citrate buffer (pH 6.0) heat-mediated retrieval typically yielding optimal results for DPYSL3 epitope accessibility . In immunocytochemistry applications, 4% paraformaldehyde fixation for 15-20 minutes at room temperature preserves DPYSL3 cytoplasmic localization while maintaining cellular morphology . For permeabilization, 0.1-0.3% Triton X-100 provides sufficient access to cytoplasmic DPYSL3 without excessive disruption of cellular structures. When using antibodies targeting the C-terminal domain (such as those recognizing AA 457-555 or AA 465-570), milder permeabilization may be preferable as these regions can be more sensitive to detergent-induced conformational changes . For dual immunofluorescence studies involving DPYSL3 and cytoskeletal elements, methanol fixation may offer advantages for preserving protein-protein interactions relevant to DPYSL3's role in cell migration.

How do post-translational modifications affect DPYSL3 antibody recognition?

Post-translational modifications (PTMs) can significantly impact DPYSL3 antibody recognition, causing unexpected variation in experimental results. DPYSL3 undergoes several PTMs including phosphorylation and SUMOylation that regulate its function in cytokine-mediated signaling pathways and cell migration . Antibodies targeting regions containing modification sites may show reduced binding when the protein is modified, yielding artificially low detection levels despite normal protein expression. For phosphorylation-sensitive applications, consider using antibodies specifically designed to recognize phosphorylated or non-phosphorylated forms, or target epitopes distant from known modification sites, such as antibodies binding the N-terminal region (AA 1-218) . When investigating DPYSL3 in signaling contexts, comparing results from multiple antibodies targeting different epitopes can help distinguish between actual protein level changes and PTM-induced detection variations. Treatment of samples with phosphatases prior to immunodetection can sometimes restore antibody recognition if phosphorylation is interfering with epitope binding.

What controls should be included when working with DPYSL3 antibodies?

Implementing appropriate controls is essential for generating reliable data with DPYSL3 antibodies. Positive tissue controls should include heart and skeletal muscle samples, where DPYSL3 is predominantly expressed . Negative controls should include tissues with minimal DPYSL3 expression or antibody diluent without primary antibody to assess non-specific binding. For quantitative applications, loading controls must be carefully selected - GAPDH may be suitable for some tissues, but in muscle samples with high metabolic activity, structural proteins like β-actin may provide more consistent normalization . When comparing DPYSL3 expression across different experimental conditions, include a standard curve of recombinant DPYSL3 protein to ensure measurements fall within the linear detection range. For genetic manipulation studies, validate antibody specificity using siRNA knockdown or CRISPR knockout controls, particularly when investigating subtle changes in DPYSL3 expression levels.

How can researchers differentiate between DPYSL3 isoforms?

Differentiating between the two reported DPYSL3 isoforms requires strategic antibody selection and experimental design. Western blotting using high-resolution SDS-PAGE (8-10% gels with extended run times) can separate the isoforms based on subtle molecular weight differences . Select antibodies targeting regions that differ between isoforms - some antibodies recognize specific amino acid sequences unique to certain isoforms. Consider using antibodies raised against the full-length protein (AA 1-570) for detecting all isoforms, while epitope-specific antibodies may preferentially recognize particular variants . RT-PCR with isoform-specific primers can complement protein detection to confirm expression patterns at the mRNA level. For definitive isoform identification, immunoprecipitation followed by mass spectrometry analysis provides the highest resolution approach, allowing precise mapping of the exact DPYSL3 variant present in your experimental system.

What are the key considerations for quantitative analysis of DPYSL3 expression?

Quantitative analysis of DPYSL3 expression demands meticulous attention to technical parameters that affect measurement accuracy. For Western blotting, establish a standard curve using recombinant DPYSL3 protein to confirm detection linearity across your expected concentration range . When comparing expression between different tissues, normalize DPYSL3 signals to total protein measurement (using Ponceau S or stain-free technology) rather than single housekeeping proteins, which can vary significantly between tissues. For ELISA-based quantification, develop standard curves with recombinant DPYSL3 prepared in the same buffer matrix as your samples to account for matrix effects . In immunohistochemistry quantification, standardize acquisition parameters including exposure times, gain settings, and thresholding criteria across all samples. When comparing DPYSL3 levels between experimental groups, process all samples simultaneously rather than in separate batches to minimize technical variation that could be misinterpreted as biological differences.

How can researchers address non-specific binding issues with DPYSL3 antibodies?

Non-specific binding with DPYSL3 antibodies can be addressed through systematic optimization of experimental conditions. If multiple bands appear in Western blots, increase antibody specificity by using more stringent washing conditions (0.1% Tween-20 in TBS with 500mM NaCl) and longer washing times . For polyclonal antibodies showing cross-reactivity, consider affinity purification against the specific antigen or switch to monoclonal antibodies with validated non-reactivity to other CRMP family proteins like CRMP-1, CRMP-2, CRMP-3, or CRMP-5 . In immunohistochemistry applications, increase blocking stringency using 5-10% normal serum from the same species as the secondary antibody, and add 0.1-0.3% Triton X-100 to reduce non-specific hydrophobic interactions. For tissues with high endogenous biotin or peroxidase activity, implement specific blocking steps using avidin/biotin blocking kits or 3% hydrogen peroxide treatment respectively. When all other approaches fail, consider testing antibodies targeting different DPYSL3 epitopes, as certain regions may be more prone to non-specific interactions in particular experimental systems.

What strategies help resolve weak or absent DPYSL3 signal issues?

When encountering weak or absent DPYSL3 signals, several optimization strategies can enhance detection sensitivity. Begin by confirming DPYSL3 expression in your sample type - remember that DPYSL3 is primarily expressed in heart and skeletal muscle, with lower expression in other tissues . For Western blotting applications, increase protein loading to 50-75 μg per lane and extend primary antibody incubation to overnight at 4°C with gentle agitation. Enhanced chemiluminescence (ECL) substrates with higher sensitivity can significantly improve signal detection. For immunohistochemistry, optimize antigen retrieval methods by testing different buffers (citrate pH 6.0 versus EDTA pH 9.0) and heating protocols . Signal amplification systems such as tyramide signal amplification (TSA) or polymer-based detection can dramatically enhance sensitivity for low-abundance DPYSL3 detection. If tissue fixation might compromise epitope accessibility, consider testing antibodies targeting different regions of DPYSL3, particularly comparing N-terminal (AA 1-218) versus C-terminal (AA 457-555) targeting antibodies, as fixation can differentially affect epitope exposure .

How should researchers interpret contradictory results from different DPYSL3 antibodies?

Contradictory results from different DPYSL3 antibodies require careful analytical approaches to resolve apparent discrepancies. First, compare the exact epitopes recognized by each antibody - antibodies targeting different regions (N-terminal AA 1-218 versus C-terminal AA 457-555) may yield different results if post-translational modifications or protein interactions affect specific domains . Verify the specificity of each antibody through knockdown/knockout validation or peptide competition assays to identify potential false positives. Consider protein conformation effects - some antibodies may preferentially recognize denatured DPYSL3 (suitable for Western blotting) while others detect native conformations (optimal for immunoprecipitation or immunohistochemistry) . If discrepancies persist, implement orthogonal detection methods such as mass spectrometry or RT-qPCR to provide independent verification of DPYSL3 presence and quantity. Multiple antibody validation is considered best practice in rigorous research - when results align between antibodies targeting different DPYSL3 epitopes, confidence in the biological significance of the findings increases substantially.

Comparison of DPYSL3 Antibodies by Target Region and Applications

Recommended Dilutions for DPYSL3 Antibody Applications