DPYSL2 Recombinant Monoclonal Antibody

What is DPYSL2 and what are its primary cellular functions?

DPYSL2, also known as CRMP2 (Collapsin Response Mediator Protein 2), is a multifunctional protein that plays central roles in nervous system development and function. It forms homo- and hetero-tetramers that facilitate neuron guidance, growth, and polarity establishment. The protein promotes microtubule assembly and is required for Sema3A-mediated growth cone collapse. Additionally, DPYSL2 plays critical roles in synaptic signaling through interactions with calcium channels. At the molecular level, DPYSL2 is involved in neurite outgrowth, axon guidance, synaptic plasticity, neuronal signaling, and neuronal survival mechanisms. Dysregulation of this protein has been implicated in various neurological disorders, including Alzheimer's disease, schizophrenia, and epilepsy .

How is DPYSL2 involved in neuronal stem cell differentiation?

DPYSL2 functions as a key regulator of neural stem cell (NSC) differentiation. Research has identified DPYSL2 as one of the differentially expressed proteins during NSC differentiation, particularly when comparing serum-alone versus PNS (peripheral nervous system) + serum treatment groups. Experimental evidence shows that overexpression of DPYSL2 in NSCs promotes differentiation toward neuronal lineages more significantly than toward astrocytic lineages. Specifically, DPYSL2 overexpression leads to a significant increase in Tuj1-positive cells (neurons) compared to control groups, with a less pronounced but still significant increase in GFAP-positive cells (astrocytes). This indicates that DPYSL2 plays a preferential role in directing NSC differentiation toward neurons, making it a potential target for neuronal regeneration strategies .

What are the structural characteristics of human DPYSL2 protein?

Human DPYSL2 is a 62.3 kDa protein consisting of 572 amino acid residues in its canonical form. The protein is primarily localized in the cytoplasm, though it also associates with membranes and the cytoskeleton. Up to two different isoforms have been reported through alternative splicing. The protein contains the sequence "PFPDFVYKRIKARSRLAELRGVPRGLYDGPVCEVSV TPKTVTPASSAKTSPAKQQAPPVRNLHQSGFSLSGAQIDDNIPRRTTQRIVAPPGGRANI TSLG" between amino acids 473-572, which has been utilized as an immunogen for antibody development. DPYSL2 belongs to the Hydantoinase/dihydropyrimidinase protein family and has been evolutionarily conserved across species, with orthologs reported in mouse, rat, bovine, frog, chimpanzee, and chicken .

How are DPYSL2 recombinant monoclonal antibodies produced?

DPYSL2 recombinant monoclonal antibodies are produced through a meticulously executed process of molecular cloning and cell culture techniques. The process begins with in vitro cloning of genes encoding both the heavy and light chains of the DPYSL2 antibody into appropriate expression vectors. These vectors are then introduced into host cells, creating an environment conducive to recombinant antibody expression within a cell culture setting. Following expression, the DPYSL2 recombinant monoclonal antibody is purified from the cell culture supernatant using affinity chromatography, which yields a highly specific antibody preparation. This recombinant approach ensures consistent production of antibodies with identical binding characteristics across different batches, providing researchers with reliable tools for DPYSL2 detection and analysis .

What are the key differences between polyclonal and recombinant monoclonal antibodies for DPYSL2 research?

Recombinant monoclonal antibodies offer several distinct advantages over polyclonal antibodies for DPYSL2 research. While polyclonal antibodies are derived from multiple B cell clones and recognize different epitopes of DPYSL2, recombinant monoclonal antibodies originate from a single clone and target a specific epitope with high specificity. This epitope specificity is crucial when investigating particular domains or post-translational modifications of DPYSL2. Furthermore, recombinant monoclonal antibodies provide consistent batch-to-batch reproducibility, eliminating the variability inherent to polyclonal antibodies. For DPYSL2 research, where distinguishing between closely related CRMP family members or specific phosphorylated forms is often necessary, the precise epitope recognition of recombinant monoclonal antibodies significantly enhances experimental reliability and interpretation .

What validation methods confirm specificity of DPYSL2 antibodies?

The specificity of DPYSL2 antibodies is confirmed through multiple validation approaches. Knockout (KO) validation represents a gold standard, where the antibody is tested against samples from DPYSL2 knockout models to confirm absence of signal. Western blot analysis demonstrates the antibody recognizes a protein of the expected molecular weight (approximately 62 kDa). Immunofluorescence studies in relevant tissues, such as mouse and rat brain sections, verify appropriate tissue and subcellular localization patterns. Cross-reactivity testing across human, mouse, and rat samples confirms species reactivity. Additionally, immunoprecipitation followed by mass spectrometry can verify the exact protein being recognized. For DPYSL2 antibodies, positive controls typically include HeLa cells, mouse/rat lung, and mouse/rat heart tissues, which express detectable levels of the target protein .

What are the optimal experimental conditions for DPYSL2 antibody applications in Western blotting?

For Western blot applications using DPYSL2 recombinant monoclonal antibodies, several parameters require optimization. The recommended dilution range is 1:500 to 1:5000, with optimal dilution determined through titration experiments for each specific antibody and sample type. Samples should be prepared in a buffer containing protease inhibitors to prevent degradation of DPYSL2, which has a calculated molecular weight of 62 kDa. When performing SDS-PAGE, 10-12% gels are typically sufficient for resolving DPYSL2. During transfer, standard PVDF or nitrocellulose membranes are suitable. Blocking should be performed with 5% non-fat dry milk or BSA in TBST, and primary antibody incubation is recommended overnight at 4°C. For detection, standard ECL systems are appropriate, with exposure times adjusted according to signal intensity. Positive control samples include HeLa cells, mouse lung, mouse heart, rat lung, and rat heart tissues, which all express detectable levels of DPYSL2 .

How should immunofluorescence protocols be optimized for DPYSL2 detection in neural tissues?

Immunofluorescence detection of DPYSL2 in neural tissues requires specific protocol optimization. The recommended antibody dilution range is 1:50 to 1:200, with the optimal concentration determined empirically for each tissue type. For tissue preparation, 4% paraformaldehyde fixation followed by either frozen sectioning or paraffin embedding is suitable, though antigen retrieval may be necessary for paraffin sections. Permeabilization with 0.1-0.3% Triton X-100 facilitates antibody access to cytoplasmic DPYSL2. Blocking with 10% normal serum from the species of the secondary antibody for 1-2 hours helps reduce background. Primary antibody incubation should be conducted overnight at 4°C, followed by appropriate fluorophore-conjugated secondary antibody incubation. DAPI counterstaining helps visualize nuclei. Confocal microscopy at 40x magnification has been successfully used to detect DPYSL2 expression in both mouse and rat brain tissues. Cytoplasmic, membrane, and cytoskeletal localization patterns should be evident in properly optimized experiments .

What considerations are important when using DPYSL2 antibodies for studying neural stem cell differentiation?

When using DPYSL2 antibodies to study neural stem cell (NSC) differentiation, several methodological considerations are crucial. First, temporal dynamics must be accounted for, as DPYSL2 expression changes significantly during differentiation processes. Sampling at multiple time points (typically days 1, 3, 7, and 14 after induction of differentiation) provides comprehensive data. Second, co-immunostaining with lineage markers such as Tuj1 (for neurons) and GFAP (for astrocytes) enables correlation between DPYSL2 expression and cell fate. Third, quantification approaches should include both the percentage of DPYSL2-positive cells and the intensity of DPYSL2 staining. Research has demonstrated that DPYSL2 overexpression promotes NSC differentiation particularly toward neuronal lineages (0.525 ± 0.02 Tuj1-positive cells in DPYSL2 overexpression group versus 0.271 ± 0.05 in control group, p<0.05). Finally, experimental manipulations of DPYSL2 expression through overexpression or knockdown approaches can establish causative relationships between DPYSL2 levels and differentiation outcomes .

How do post-translational modifications of DPYSL2 affect antibody recognition?

Post-translational modifications (PTMs) significantly impact DPYSL2 antibody recognition and have crucial biological implications. DPYSL2 undergoes extensive phosphorylation, particularly by kinases including GSK3β, CDK5, and ROCK, which regulate its function in neurite outgrowth and axon guidance. Hyperphosphorylation of DPYSL2 has been implicated in Alzheimer's disease pathogenesis. When selecting antibodies, researchers must determine whether they need antibodies that recognize total DPYSL2 regardless of phosphorylation state, or phospho-specific antibodies that detect particular phosphorylated residues. Antibodies raised against sequences containing phosphorylation sites may show differential binding depending on the phosphorylation status. For comprehensive analysis, complementary approaches using both total and phospho-specific antibodies are recommended. Additionally, treatment with phosphatases before immunodetection can help distinguish whether observed changes in DPYSL2 detection are due to altered expression or modified phosphorylation states .

What strategies enable distinction between DPYSL2 and other CRMP family members?

Distinguishing DPYSL2 (CRMP2) from other CRMP family members (CRMP1, CRMP3, CRMP4, and CRMP5) requires careful antibody selection and experimental design. Despite sharing approximately 50-75% sequence homology, these proteins have distinct functions in neuronal development. For antibody-based discrimination, selecting antibodies raised against regions with maximal sequence divergence is essential. The C-terminal region (amino acids 473-572) contains distinctive sequences often used for generating DPYSL2-specific antibodies. Western blotting can distinguish family members partly by molecular weight differences: DPYSL2 (62 kDa), CRMP1 (62 kDa), CRMP3 (64 kDa), CRMP4 (65 kDa), and CRMP5 (68 kDa). Isoelectric focusing combined with Western blotting offers additional separation based on differing isoelectric points. For transcriptional analysis, designing PCR primers specifically in non-homologous regions ensures amplification of only the target CRMP. When possible, validation experiments should include positive controls for each CRMP family member to confirm specificity .

How can DPYSL2 antibodies be effectively employed in studying neurodegenerative diseases?

DPYSL2 antibodies provide valuable tools for investigating neurodegenerative mechanisms, particularly in Alzheimer's disease where DPYSL2 hyperphosphorylation appears to play a pathogenic role. When studying neurodegenerative conditions, researchers should employ a multi-faceted approach. First, analyzing both total and phosphorylated DPYSL2 levels in patient-derived tissues or appropriate disease models is essential, as changes in phosphorylation often exceed changes in total protein levels. Second, co-immunoprecipitation studies using DPYSL2 antibodies can reveal altered protein interactions in disease states, such as disrupted binding to tubulin or increased association with tau. Third, immunohistochemistry in brain sections can demonstrate altered subcellular localization or inclusion formation. Fourth, longitudinal studies in animal models using DPYSL2 antibodies can track progressive changes during disease development. Finally, therapeutic intervention studies targeting DPYSL2 phosphorylation should incorporate antibodies that specifically recognize the relevant phosphorylation sites to monitor treatment efficacy .

What are the common sources of background and non-specific binding when using DPYSL2 antibodies?

Non-specific binding and background issues with DPYSL2 antibodies can arise from several sources that require systematic troubleshooting. Cross-reactivity with other CRMP family members represents a primary concern due to sequence homology. This can be addressed by using antibodies raised against unique epitopes and validating with known positive and negative controls. Secondary antibody cross-reactivity, particularly in multi-labeling experiments, can be minimized by using highly cross-adsorbed secondary antibodies and implementing appropriate blocking steps. Endogenous peroxidase or phosphatase activity in tissues can generate false-positive signals in enzyme-based detection systems; this requires quenching steps with H₂O₂ or levamisole, respectively. Autofluorescence, especially in neural tissues with lipofuscin, can be reduced using Sudan Black B treatment or spectral unmixing during confocal microscopy. Finally, overfixation of tissues may mask epitopes; titrating fixation conditions and implementing appropriate antigen retrieval methods can restore antibody accessibility to DPYSL2 epitopes .

How can researchers optimize co-immunoprecipitation experiments with DPYSL2 antibodies?

Successful co-immunoprecipitation (co-IP) of DPYSL2 and its interaction partners requires optimization of several parameters. Lysis buffer composition is critical—mild non-ionic detergents like NP-40 or Triton X-100 (0.5-1%) preserve protein interactions, while phosphatase inhibitors maintain phosphorylation-dependent interactions. Pre-clearing lysates with protein A/G beads reduces non-specific binding. For the antibody-antigen interaction, using 2-5 μg of DPYSL2 antibody per 500 μg of total protein typically yields good results. Incubation should occur overnight at 4°C with gentle rotation to preserve fragile protein complexes. When investigating interactions with cytoskeletal components, adding stabilizing agents like taxol (for microtubules) or phalloidin (for actin) to the lysis buffer can preserve these associations. For detecting transient interactions, chemical crosslinking prior to lysis using membrane-permeable crosslinkers like DSP (dithiobis(succinimidyl propionate)) may be necessary. Finally, validation of co-IP results using reciprocal co-IP (using antibodies against the interaction partner) provides stronger evidence for genuine protein-protein interactions .

What controls should be included when quantifying DPYSL2 expression in comparative studies?

Robust quantification of DPYSL2 expression in comparative studies requires implementation of several control measures. Loading controls are essential: for Western blotting, housekeeping proteins like GAPDH, β-actin, or α-tubulin should be used, while normalizing to total protein loading (assessed by Ponceau S or similar stains) provides an alternative approach less susceptible to experimental manipulations. Calibration curves using purified recombinant DPYSL2 protein enable absolute quantification rather than relative comparisons. For immunohistochemistry/immunofluorescence studies, standardized image acquisition parameters and systematic sampling approaches prevent selection bias. Technical replicates (minimum of three) account for methodological variation, while biological replicates address sample-to-sample variability. For experiments manipulating DPYSL2 expression, both positive controls (known to increase DPYSL2) and negative controls (known to decrease DPYSL2) provide reference points. When comparing phosphorylated and total DPYSL2, analyzing the phospho-to-total ratio rather than absolute values often provides more meaningful biological insights .

How should researchers analyze DPYSL2 expression across different neural cell populations?

Analyzing DPYSL2 expression across diverse neural cell populations requires integrating multiple analytical approaches. Flow cytometry offers quantitative single-cell analysis when combined with permeabilization protocols suitable for detecting cytoplasmic DPYSL2 alongside surface markers of neural subtypes. Fluorescence-activated cell sorting (FACS) followed by Western blotting or qPCR enables examination of DPYSL2 expression in isolated neural subpopulations. For tissue sections, multiplexed immunofluorescence combining DPYSL2 antibodies with markers for neurons (NeuN, Tuj1), astrocytes (GFAP), oligodendrocytes (MBP, Olig2), and neural stem cells (Nestin, Sox2) reveals cell type-specific expression patterns. Laser capture microdissection coupled with proteomics or transcriptomics provides region-specific analysis. Single-cell RNA sequencing data can be mined to examine DPYSL2 expression variability across neural subtypes. Quantitative analysis should include both the percentage of DPYSL2-positive cells within each population and the intensity of expression, as both metrics provide complementary information about regulation patterns .

What bioinformatic approaches help interpret DPYSL2 antibody-based proteomics data?

Bioinformatic analysis enhances interpretation of DPYSL2 antibody-based proteomics data by revealing functional networks and regulatory mechanisms. Pathway enrichment analysis using databases like KEGG, Reactome, or GO identifies biological processes associated with DPYSL2 and its interacting partners. Protein-protein interaction network analysis through tools like STRING (9606.ENSP00000309539) places DPYSL2 in its functional context, with visualization software like Cytoscape enabling graphical representation of these networks. Motif analysis around phosphorylation sites recognized by phospho-specific antibodies can reveal regulatory kinases. Comparative analysis across species leveraging orthologous relationships (with DPYSL2 orthologs identified in mouse, rat, bovine, frog, chimpanzee, and chicken) highlights evolutionarily conserved functions. Integration of antibody-derived proteomics data with transcriptomics datasets through correlation analysis can identify potential transcriptional regulators of DPYSL2. When analyzing disease-related changes, comparison with existing databases of disease-associated proteins helps contextualize findings within broader pathological mechanisms .

How can contradictory results between different DPYSL2 antibodies be reconciled?

Contradictory results between different DPYSL2 antibodies represent a significant research challenge requiring systematic investigation. Epitope mapping should be performed to determine precisely which regions of DPYSL2 each antibody recognizes, as differences may reflect epitope availability rather than actual protein abundance. Post-translational modifications, particularly phosphorylation, may mask epitopes recognized by certain antibodies while leaving others unaffected. Alternative splicing of DPYSL2 can generate isoforms lacking specific epitopes, leading to discrepant results between antibodies targeting different regions. Cross-reactivity with other CRMP family members should be evaluated through controls using recombinant proteins. Methodological differences in sample preparation (extraction buffers, fixation protocols) may differentially expose epitopes. For definitive resolution, orthogonal techniques such as mass spectrometry can provide antibody-independent protein identification and quantification. When publishing results, transparent reporting of the specific antibody used (including catalog number and lot), experimental conditions, and observed molecular weight is essential for result interpretation and reproducibility .