CNDP2 Antibody

What is CNDP2 and why is it significant for research?

CNDP2 (carnosinase-2 or CN2) is a cytosolic non-specific dipeptidase belonging to the peptidase M20A family that catalyzes peptide bond hydrolysis in dipeptides . It plays crucial roles in regulating peptide metabolism across various tissues and has significant implications in cancer research, with context-dependent expression patterns across different cancer types . CNDP2 has a calculated molecular weight of 53 kDa, though observed molecular weights in experiments typically range from 44-53 kDa, likely due to post-translational modifications or isoform variations .

What applications are CNDP2 antibodies validated for?

CNDP2 antibodies have been validated for multiple experimental applications, with specific reactivity against human, mouse, and rat samples. The following table summarizes common applications:

Researchers should verify specific validation data for their antibody of interest .

What are the recommended dilutions for CNDP2 antibodies?

Optimal antibody dilutions vary by application and specific antibody. For polyclonal antibodies like Proteintech's 14925-1-AP, the following dilutions are recommended:

It's essential to optimize these dilutions for specific experimental conditions and sample types, as results may be sample-dependent .

How should CNDP2 antibodies be stored for maximum stability?

CNDP2 antibodies should be stored at -20°C, where they typically remain stable for one year after shipment. Many commercial preparations come in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain antibody stability. Notably, for -20°C storage, aliquoting is often unnecessary, simplifying laboratory handling. Some preparations (particularly smaller sizes like 20μl) may contain 0.1% BSA as a stabilizer .

How can researchers validate the specificity of CNDP2 antibodies?

Confirming antibody specificity is crucial for reliable results. Researchers should consider these validation approaches:

• Positive control testing: Verify reactivity in samples known to express CNDP2, such as HepG2 cells, LNCaP cells, or kidney tissue from mice or rats .

• Knockdown validation: Compare staining between wild-type cells and CNDP2 knockdown cells. Several RNAi target sequences have been validated, including 5′-ACT TTG ACA TAG AGG AGT T-3′ .

• Cross-reactivity assessment: If working across species, validate reactivity in each target species. Some antibodies show reactivity with human, mouse, and rat samples, with cited reactivity for zebrafish .

• Multiple antibody comparison: Use alternative antibodies targeting different epitopes to confirm similar staining patterns.

• Western blot analysis: Verify that the observed molecular weight matches the expected size (44-53 kDa) .

What sample preparation methods enhance CNDP2 detection?

Optimal sample preparation varies by application:

• For IHC: Antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may serve as an alternative . Formalin-fixed, paraffin-embedded tissues typically require more rigorous antigen retrieval than frozen sections.

• For Western blot: Standard protein extraction protocols are suitable, with particular success reported using lysates from HepG2 cells, LNCaP cells, and kidney tissue .

• For IP: Use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate, with demonstrated success in mouse testis tissue .

• For IF: Standard fixation (typically 4% paraformaldehyde) and permeabilization protocols for cytosolic proteins work well, with successful detection reported in HeLa cells .

How can researchers troubleshoot weak or absent CNDP2 signals?

When faced with detection challenges, consider these methodological adjustments:

• Antibody concentration: Increase primary antibody concentration within the recommended range.

• Antigen retrieval optimization: For IHC, test both TE buffer pH 9.0 and citrate buffer pH 6.0, and adjust retrieval time and temperature.

• Incubation parameters: Extend primary antibody incubation time or adjust temperature (4°C overnight often improves sensitivity).

• Expression verification: Confirm CNDP2 expression in your sample type; expression levels vary significantly between normal and cancer tissues .

• Detection system enhancement: Use a more sensitive detection system (e.g., polymer-based systems for IHC, enhanced chemiluminescence for WB).

• Blocking optimization: Adjust blocking conditions to reduce background while preserving specific signal.

What is known about CNDP2's role in cancer biology?

CNDP2 exhibits intriguing context-dependent roles across different cancer types:

In colon cancer, CNDP2 expression is markedly higher in tumor tissues (50.7% high expression) compared to normal colon mucosa (10.3% high expression) . Knockdown experiments demonstrate that CNDP2 knockdown blocks cell cycle progression, with increased G2/M phase fraction and reduced S phase proportion .

Contrastingly, in gastric cancer, CNDP2 reintroduction transcriptionally upregulates p38 and activates c-Jun NH2-terminal kinase (JNK), while its loss increases ERK phosphorylation, suggesting pathway-specific mechanisms for its tumor suppressor function .

How can researchers design effective CNDP2 knockdown experiments?

For successful CNDP2 functional studies through gene silencing:

• RNAi approach: Multiple validated target sequences exist, with 5′-ACT TTG ACA TAG AGG AGT T-3′ demonstrating high knockdown efficiency . These can be cloned into lentiviral vectors (e.g., GV112 with hU6-MCS-CMV-Puromycin) for stable expression.

• CRISPR/Cas9 system: For complete knockout, gRNA design tools can identify effective target sequences. In Drosophila studies, sequences such as 5′-GUAAAAUAGAUUCGACGUAA-3′ and 5′-GAACCAGAUAUGACCCGCGA-3′ have been successfully employed .

• Experimental validation: Confirm knockdown efficiency at both mRNA level (RT-PCR, qPCR with primers targeting CNDP2) and protein level (Western blot with validated antibodies) .

• Functional assays: Assess proliferation, colony formation, cell cycle distribution, and in vivo tumor growth in xenograft models to comprehensively evaluate CNDP2's functional impact .

How does CNDP2 affect cellular metabolism and signaling pathways?

CNDP2 plays a critical role in cellular metabolism with far-reaching consequences:

• Metabolic impact: CNDP2 knockout in human proximal tubule cells results in:

  • Accumulation of cellular dipeptides

  • Reduction of amino acids

  • Imbalance of related metabolic pathways

  • Disruption of energy supply

• Signaling pathway interactions: CNDP2 interfaces with multiple signaling networks:

  • In gastric cancer, it modulates the MAPK pathway, with reintroduction upregulating p38 and activating JNK

  • Loss of CNDP2 increases ERK phosphorylation

  • In colon cancer, it interacts with EGFR, cyclin B1, and cyclin E pathways

• Cellular function effects: CNDP2 knockout affects:

  • Cell viability and proliferation

  • Ion and macromolecule transport via trans- and paracellular pathways

  • Regulatory and transport protein abundance

RNA-seq analyses of CNDP2-knockout cells reveal altered protein metabolism and ion transport, highlighting its multifaceted role in cellular homeostasis .

What model systems are available for studying CNDP2 function?

Researchers have several options for investigating CNDP2 in model systems:

• Cell culture models: Multiple cell lines have been characterized for CNDP2 expression, including:

  • Colon cancer lines (RKO, HCT116, DLD-1, LS174T, HT29, SW480, LoVo) with high expression

  • Normal colon epithelial cells (FHC) with low expression

  • Various cancer and normal cell types for comparative studies

• Animal models:

  • Drosophila melanogaster: Null dCNDP2 mutants and transgenic lines for inducible expression have been generated

  • Mouse models: CNDP2 antibodies show reactivity with mouse tissues, facilitating in vivo studies

  • Xenograft models: CNDP2-modified cancer cells can be studied in nude mice to evaluate tumor growth and progression

  • Zebrafish: Cited reactivity for CNDP2 antibodies suggests potential for developmental studies

• Human tissue samples: CNDP2 expression has been characterized in various human tissues, including:

  • Normal vs. cancerous colon tissues

  • Gastric cancer specimens

  • Hepatocellular carcinoma samples

  • Proximal tubular cells

Each model system offers unique advantages for investigating specific aspects of CNDP2 biology.

How can researchers investigate discrepancies in CNDP2 function between cancer types?

The contradictory roles of CNDP2 across cancer types present intriguing research opportunities:

• Isoform-specific studies: The specific isoform lacking exons 3 and 4 is present in all fetal tissues but only adult liver . Researchers should investigate whether differential isoform expression explains functional discrepancies between cancer types.

• Tissue-specific signaling context: Compare CNDP2's interaction with MAPK pathway components (p38, JNK, ERK) across different tissue types to determine how cellular context influences its function .

• Metabolomic profiling: Since CNDP2 affects dipeptide and amino acid metabolism, comprehensive metabolomic analysis of different cancer types might reveal tissue-specific metabolic requirements that explain CNDP2's divergent roles .

• Protein interaction networks: Immunoprecipitation followed by mass spectrometry could identify tissue-specific binding partners that modify CNDP2's function across cancer types.

• In vivo modeling: Develop conditional tissue-specific CNDP2 knockout models to compare its function across multiple organs simultaneously in the same genetic background.

What techniques are recommended for studying CNDP2's role in kidney function?

CNDP2 is abundant in human proximal tubules with significant implications for kidney physiology:

• Cellular models: Establish CNDP2-knockout in human proximal tubule cells using CRISPR/Cas9 technology for detailed functional studies .

• Transport assays: Measure paracellular permeability and ion transport in kidney cell models with modulated CNDP2 expression .

• Metabolomic analysis: Characterize dipeptide and amino acid profiles in kidney tissues and cells with altered CNDP2 expression .

• RNA-seq approach: Analyze transcriptional changes following CNDP2 modulation to identify affected pathways in kidney cells .

• In vivo studies: Examine the relevance of CNDP2 for nephron function and regulation of body homeostasis in appropriate animal models .

How can researchers better characterize CNDP2's enzymatic activity?

For comprehensive enzymatic characterization:

• Substrate specificity: Determine CNDP2's activity against various dipeptides, including carnosine (β-Ala-His), Ala-Gln, and Tyr-Asp, which have demonstrated biological functions .

• Kinetic analysis: Measure reaction rates under varying substrate concentrations to determine Km and Vmax parameters.

• Inhibitor studies: Identify specific inhibitors of CNDP2 activity for functional studies and potential therapeutic development.

• Structure-function analysis: Compare enzymatic activities of different CNDP2 isoforms to understand the functional significance of alternative splicing.

• Cellular context: Evaluate how the cellular environment (pH, ion concentrations, metabolic state) affects CNDP2's enzymatic activity.