DPYSL5 Antibody

What antigen retrieval methods are recommended for DPYSL5 immunohistochemistry?

For immunohistochemical detection of DPYSL5, the suggested antigen retrieval method involves using TE buffer at pH 9.0 . Alternatively, antigen retrieval may be performed with citrate buffer at pH 6.0. The recommended dilution range for IHC applications is 1:50-1:500 . When working with mouse brain tissue, which has demonstrated positive IHC detection with DPYSL5 antibody, it is crucial to optimize the antigen retrieval conditions to ensure specific binding while minimizing background staining.

What cell lines and tissue samples are validated for DPYSL5 antibody applications?

DPYSL5 antibody has been validated in several experimental systems:

• Positive Western blot detection in mouse and rat brain tissue

• Positive immunoprecipitation in mouse brain tissue

• Positive immunohistochemistry in mouse brain tissue

• Positive immunofluorescence/ICC in SH-SY5Y cells

For research on prostate cancer models, DPYSL5 antibody has been used successfully to detect overexpression in treatment-induced neuroendocrine prostate cancer samples .

How can I optimize DPYSL5 antibody for detecting neuroendocrine markers in prostate cancer research?

When studying DPYSL5 in prostate cancer, particularly treatment-induced neuroendocrine prostate cancer (t-NEPC), consider the following optimization approach:

• Sample preparation: Use freshly prepared tissue lysates or properly fixed tissue sections from treatment-resistant tumors, as DPYSL5 is expressed in 40% of t-NEPC-like patient tumors with strong or moderate intensity (immunoscores 2/3 or 3/3) .

• Co-staining strategy: Implement a multiplex immunohistochemistry approach with DPYSL5 antibody alongside other neuroendocrine markers (SYP, CGA, NCAM) and AR markers to create a comprehensive profile. DPYSL5 significantly correlates with SYP (Pearson 0.4927), CGA (Pearson 0.5364), and NCAM (Pearson 0.6121) while inversely correlating with AR (Pearson −0.428) and PSA (Pearson −0.3412) .

• Quantification method: Develop a scoring system based on the correlation data presented in this table:

This quantification approach will provide robust statistical support for your findings when analyzing DPYSL5 expression in relation to other relevant markers in prostate cancer progression .

What are the technical considerations for using DPYSL5 antibody in co-immunoprecipitation experiments studying protein-protein interactions?

When designing co-immunoprecipitation (Co-IP) experiments with DPYSL5 antibody to investigate protein-protein interactions:

• Input optimization: Use 0.5-4.0 μg of DPYSL5 antibody for 1.0-3.0 mg of total protein lysate, as recommended for standard IP applications . For brain tissue samples, which show positive IP detection, adjust the antibody amount based on the abundance of DPYSL5 in your specific samples.

• Lysis buffer selection: Choose a lysis buffer that maintains native protein conformation while efficiently extracting DPYSL5 and its interacting partners. Since DPYSL5 is involved in EZH2-mediated PRC2 activation , consider using buffers that preserve nuclear protein interactions when studying these pathways.

• Controls implementation: Include the following controls:

  • Negative control: IgG from the same species as the DPYSL5 antibody

  • Input control: 5-10% of the lysate used for IP

  • Reciprocal IP: If studying a specific interaction, perform reverse IP with antibodies against the potential interacting partners

• Detection strategy: Use specific antibodies against suspected interacting proteins in Western blot analysis of the immunoprecipitated complex. For prostate cancer research, consider probing for EZH2 and components of the PRC2 complex, as DPYSL5 overexpression leads to upregulation of EZH2 protein levels and increased H3K27 trimethylation .

How should conflicting DPYSL5 expression data between immunohistochemistry and Western blot be interpreted in heterogeneous tumor samples?

When encountering discrepancies between DPYSL5 detection methods in heterogeneous tumor samples:

• Spatial heterogeneity assessment: Perform multiple IHC sections across different regions of the tumor to map DPYSL5 expression patterns. In t-NEPC patient tumors, DPYSL5 shows heterogeneous expression with 40% of tumors showing strong or moderate intensity .

• Sample preparation differences: Consider that Western blot analysis uses whole tissue lysates that may dilute focal high expression, while IHC preserves spatial information. For tumors undergoing neuroendocrine transformation, this spatial heterogeneity is particularly important.

• Quantitative comparison approach:

  • For IHC: Use an immunoscore system (0-3) as implemented in the CRPC-t-NEPC-like tissue microarray analysis

  • For Western blot: Normalize DPYSL5 signal to housekeeping proteins and compare relative expression

• Biological interpretation: Integrate findings with clinical parameters and expression of related markers. Since DPYSL5 expression significantly correlates with neuroendocrine markers and inversely with AR and PSA , contextualize discrepancies within the broader molecular profile of each sample.

• Resolution strategy: If discrepancies persist, validate with a third method such as RT-qPCR for DPYSL5 mRNA expression or use a different antibody that recognizes a distinct epitope of DPYSL5.

What experimental design is optimal for investigating DPYSL5's role in neuronal-like phenotype induction in cancer cell models?

To investigate DPYSL5's role in inducing neuronal-like phenotypes in cancer models:

• Genetic manipulation approach:

  • Overexpression system: Use lentiviral or plasmid vectors to overexpress DPYSL5 in prostate cancer cell lines (e.g., LNCaP, C42B)

  • Knockdown system: Implement siRNA or shRNA targeting DPYSL5 in cells with high endogenous expression

  • CRISPR-Cas9: Consider gene editing for complete knockout studies or promoter modification

• Phenotypic assessment:

  • Morphological analysis: Quantify neurite-like extensions and cell body changes using phase-contrast microscopy and neuron-specific staining

  • Invasion assay: Implement spheroid invasion assays using Matrigel with live-cell imaging to monitor invasive behavior over time (e.g., every 6 hours for 4 days)

  • Proliferation monitoring: Track spheroid growth rates, as DPYSL5 overexpressing spheroids grow significantly faster than control spheroids

• Molecular characterization:

  • Neuronal marker panel: Assess expression of neuronal lineage markers (NSE, CGA, ASCL1) by qRT-PCR and Western blot

  • Invasion markers: Measure protein levels of invasion-inducing factors like Snail

  • Stemness evaluation: Analyze stemness markers associated with DPYSL5 overexpression

• Functional validation:

  • Drug response testing: Evaluate sensitivity to AR inhibitors like Enzalutamide in DPYSL5-modified cells

  • Cell cycle analysis: Assess cell cycle distribution changes, particularly G1 phase arrest patterns

  • In vivo models: Consider chick chorioallantoic membrane (CAM) tumors to validate in vitro findings

What are common causes of non-specific binding when using DPYSL5 antibody, and how can they be addressed?

When encountering non-specific binding with DPYSL5 antibody:

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, normal serum)

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Use blocking agent in both primary and secondary antibody dilutions

• Antibody dilution adjustment:

  • Use the upper end of the recommended dilution range (e.g., 1:12000 for WB, 1:500 for IHC/IF)

  • Perform a titration series to identify the optimal concentration for your specific sample

• Washing protocol enhancement:

  • Increase the number of washing steps (5-6 washes instead of 3)

  • Extend washing time (10-15 minutes per wash)

  • Add low concentrations of detergent (0.05-0.1% Tween-20) to wash buffers

• Sample-specific considerations:

  • For brain tissue: Use fresh tissue samples and optimize fixation times

  • For cell lines: Ensure high specificity validation in SH-SY5Y cells, which show positive IF/ICC detection

• Cross-reactivity elimination:

  • Pre-absorb the antibody with the immunizing peptide if available

  • Use tissue from DPYSL5 knockout models as negative controls if accessible

How can DPYSL5 antibody be validated for specificity in novel experimental systems?

To validate DPYSL5 antibody specificity in new experimental systems:

• Molecular weight confirmation:

  • Verify that the detected band corresponds to the expected molecular weight of 61 kDa

  • Look for post-translational modifications that might alter the observed molecular weight

• Genetic manipulation controls:

  • Overexpression: Transfect cells with DPYSL5 expression vector and confirm increased signal

  • Knockdown: Use siRNA/shRNA targeting DPYSL5 and verify reduced signal

  • Use both approaches to establish a dynamic range of detection

• Peptide competition assay:

  • Pre-incubate antibody with the immunizing peptide (if available)

  • Compare signal between blocked and unblocked antibody applications

• Multiple antibody validation:

  • Use alternative antibodies targeting different epitopes of DPYSL5

  • Compare staining patterns across different antibodies

• Tissue/cell type positive controls:

  • Include known positive samples (mouse brain tissue, rat brain tissue, SH-SY5Y cells)

  • For prostate cancer research, use t-NEPC patient tumors with confirmed high DPYSL5 expression

How can DPYSL5 antibody be integrated into multiplex immunofluorescence panels to study lineage plasticity in cancer progression?

For multiplex immunofluorescence incorporating DPYSL5 antibody:

• Panel design strategy:

  • Core markers: DPYSL5 (neural development), AR (androgen receptor signaling), SYP/CGA/NCAM (neuroendocrine markers)

  • Additional markers: EZH2 (epigenetic regulation), cell cycle proteins, stemness markers

  • Panel organization based on correlation strengths identified in t-NEPC research

• Technical parameters:

  • Antibody compatibility: Test for cross-reactivity between primary antibodies

  • Fluorophore selection: Choose fluorophores with minimal spectral overlap

  • Antibody order: Apply DPYSL5 antibody at the optimal stage in the sequential staining protocol

• Spatial analysis approach:

  • Quantify co-localization coefficients between DPYSL5 and other markers

  • Implement neighborhood analysis to identify spatial relationships in heterogeneous tumors

  • Correlate DPYSL5 expression patterns with morphological features

• Clinical correlation:

  • Link multiplex data to patient outcomes using survival analysis

  • Patients with high DPYSL5 expression show significantly lower disease-free survival compared to those with low expression

  • Establish DPYSL5 expression thresholds for prognostic stratification

What considerations are important when designing time-course experiments to study DPYSL5 dynamics during treatment-induced neuroendocrine differentiation?

For time-course experiments investigating DPYSL5 in treatment-induced neuroendocrine differentiation:

• Experimental timeline design:

  • Early time points: 24h, 48h, 72h after treatment initiation

  • Intermediate points: 1 week, 2 weeks

  • Late time points: 4 weeks, 8 weeks, 12 weeks

  • Include recovery periods after treatment withdrawal

• Treatment protocol:

  • Use clinically relevant AR inhibitors like Enzalutamide (ENZ)

  • Implement dosing strategies that mimic clinical protocols

  • Include combination treatments relevant to clinical practice

• Sampling considerations:

  • Harvest matched samples for multiple analyses (protein, RNA, morphology)

  • Include biological replicates at each time point

  • Maintain consistent sampling procedures throughout the time course

• Analytical approach:

  • Track DPYSL5 expression in relation to AR activity

  • Monitor the emergence of neuronal lineage markers (NSE, CGA, ASCL1)

  • Assess EZH2 levels and H3K27 trimethylation status

  • Evaluate morphological changes using quantitative imaging metrics

• Mechanistic validation:

  • Implement DPYSL5 knockdown at critical time points to assess reversibility

  • Evaluate the impact of DPYSL5 depletion on ENZ-reduced cell proliferation and cell cycle arrest

  • Correlate changes in DPYSL5 with alterations in EZH2 and truncated JARID2 protein levels

How might the latest research on DPYSL5 inform future antibody-based therapeutic approaches for neuroendocrine cancers?

Recent research on DPYSL5 suggests several potential directions for antibody-based therapeutic approaches:

• Target validation considerations:

  • DPYSL5 is significantly upregulated in AR-negative treatment-induced neuroendocrine tumors

  • Its expression correlates with poor disease-free survival

  • DPYSL5 promotes cancer cell plasticity via EZH2-mediated PRC2 activation

• Therapeutic antibody development strategy:

  • Design antibodies that specifically target DPYSL5-expressing cancer cells

  • Develop antibody-drug conjugates delivering cytotoxic payloads to DPYSL5-positive cells

  • Consider bispecific antibodies targeting DPYSL5 and other neuroendocrine markers

• Combination therapy approach:

  • Pair DPYSL5-targeting antibodies with EZH2 inhibitors

  • Investigate synergy with conventional treatments for neuroendocrine tumors

  • Test efficacy in treatment-resistant models where DPYSL5 is highly expressed

• Biomarker utilization:

  • Use DPYSL5 antibodies for patient stratification in clinical trials

  • Develop companion diagnostics to identify patients likely to respond to DPYSL5-targeted therapies

  • Monitor treatment response using DPYSL5 expression dynamics

• Practical limitations to address:

  • Tumor heterogeneity and variable DPYSL5 expression within patients

  • Potential for resistance mechanisms through alternative pathways

  • Need for highly specific antibodies to avoid off-target effects in normal neural tissues

By integrating these considerations, researchers can leverage the growing understanding of DPYSL5 biology to develop novel therapeutic strategies for addressing the challenges of treatment-resistant neuroendocrine malignancies.