NAALADL2 Antibody

What is NAALADL2 and what are its key structural characteristics?

NAALADL2 (N-acetylated alpha-linked acidic dipeptidase like 2) is a member of the peptidase M28 family of enzymes. It functions as a type II transmembrane protein with potential O-glycosyl hydrolase activity. The human NAALADL2 protein is 795 amino acids in length and contains several distinct structural domains:

• N-terminal cytoplasmic segment

• Transmembrane domain

• Peptidase domain (amino acids 444-596)

• TfR-like dimerization region (amino acids 688-777)

Multiple splice variants have been identified, including forms with alternative start sites at Met18 and Met283, as well as various deletions and substitutions in the C-terminal region . Human NAALADL2 shares high sequence homology with other species - approximately 87% amino acid sequence identity with mouse NAALADL2 and 82% with canine NAALADL2 over amino acids 152-795 .

What tissue expression patterns have been documented for NAALADL2?

NAALADL2 has been detected in multiple human tissues, with particularly notable expression in:

• Kidney: Immunohistochemistry studies have revealed specific localization to the cytoplasm of epithelial cells in convoluted tubules of the kidney .

• Prostate: Flow cytometry analysis has confirmed NAALADL2 expression in LNCaP human prostate cancer cell lines .

• Lymphatic tissues: NAALADL2-AS2 (antisense RNA) has been documented in diffuse large B-cell lymphoma (DLBCL) tissues, with expression in both cytoplasm and nucleus of DLBCL cells (U-2932 and OCI-Ly19) .

When designing tissue-specific experiments, researchers should account for these differential expression patterns and validate antibody specificity in their tissue of interest.

What applications are NAALADL2 antibodies validated for?

NAALADL2 antibodies have been validated for multiple experimental applications with specific optimization parameters:

For optimal results, it is advisable to conduct preliminary titration experiments with your specific samples and validate the antibody performance using appropriate positive and negative controls.

How can I distinguish between different NAALADL2 splice variants in my experiments?

Distinguishing between NAALADL2 splice variants requires careful selection of antibodies targeting specific epitopes unique to each variant. Based on current research:

• Epitope mapping strategy: Select antibodies raised against regions that differ between splice variants. For instance, an antibody recognizing the region between amino acids 292-795 would not detect the splice variant with the 4 amino acid substitution in this region .

• Western blot analysis: Multiple bands may appear when analyzing NAALADL2 expression. Variants will display different molecular weights:

  • Full-length NAALADL2: ~87 kDa

  • Variant with Met18 alternative start: ~85 kDa

  • Variant with Met283 alternative start: ~56 kDa

• RT-PCR verification: Design primers spanning junction regions specific to each splice variant to confirm expression at the mRNA level before performing protein analysis.

• Combined approaches: Use a panel of antibodies targeting different epitopes, combined with molecular techniques, to fully characterize the splice variant profile in your experimental system.

What controls should be implemented when using NAALADL2 antibodies for NAALADL2-AS2 studies?

When investigating the relationship between NAALADL2 protein and its antisense RNA (NAALADL2-AS2), several critical controls should be implemented:

• RNA-protein distinction controls: Since NAALADL2-AS2 is an RNA molecule while NAALADL2 is a protein, experiments must clearly distinguish between these molecules:

  • Include RNase treatment controls for RNA visualization experiments

  • Include protease treatment controls for protein visualization experiments

• Co-localization validation: When examining potential interactions between NAALADL2 and NAALADL2-AS2:

  • Use RNA fluorescence in situ hybridization (FISH) for NAALADL2-AS2 detection

  • Employ immunofluorescence for NAALADL2 protein detection

  • Include appropriate negative controls (non-DLBCL tissues)

• Expression correlation analysis: When analyzing regulatory relationships:

  • Include siRNA knockdown of NAALADL2-AS2 to assess effects on NAALADL2 protein

  • Include overexpression systems to evaluate reciprocal effects

How does NAALADL2-AS2 function as a competing endogenous RNA in DLBCL, and what are the methodological approaches to study this mechanism?

NAALADL2-AS2 functions as a competing endogenous RNA (ceRNA) in diffuse large B-cell lymphoma through a complex regulatory network:

• Mechanistic pathway: NAALADL2-AS2 acts by sequestering specific microRNAs, particularly miR-34a and miR-125a, preventing them from binding their target mRNAs. This leads to upregulation of BCL-2, promoting cell survival and drug resistance in DLBCL cells .

• Experimental approaches to investigate this mechanism:

  a) RNA-FISH for localization:

  • Fix cells in 4% paraformaldehyde (15 minutes)

  • Permeabilize with 0.5% Triton X-100 (15 minutes at 4°C)

  • Hybridize with digoxigenin-labeled probes at 55°C for 4 hours

  • Counterstain with DAPI for nuclear visualization

  b) Gene knockdown studies:

  • Transfect DLBCL cells with NAALADL2-AS2 siRNA

  • Validate knockdown efficiency by qPCR

  • Assess effects on miRNA levels (miR-34a, miR-125a)

  • Measure BCL-2 expression changes by western blotting

  c) Luciferase reporter assays:

  • Construct reporter vectors containing predicted binding sites

  • Co-transfect with miRNA mimics or inhibitors

  • Measure luciferase activity to confirm direct interactions

• Functional assessment:

  • Cell proliferation evaluation using BrdU assays

  • Apoptosis quantification using Annexin V-FITC/PI staining

  • Drug sensitivity testing using ATP-TCA experiments with varying concentrations of doxorubicin and rituximab

What are the critical factors for optimizing immunohistochemistry protocols with NAALADL2 antibodies?

Successful immunohistochemistry with NAALADL2 antibodies requires optimization of several key parameters:

• Antigen retrieval method: Heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic has proven effective for NAALADL2 detection in paraffin-embedded tissues . Compare multiple retrieval methods:

  • Heat-induced (citrate buffer, pH 6.0)

  • Heat-induced (EDTA buffer, pH 9.0)

  • Enzymatic retrieval (proteinase K)

• Antibody concentration optimization:

  • Initial recommended dilution range: 1:50 - 1:200

  • Perform titration series to determine optimal signal-to-noise ratio

  • Include positive control tissues (kidney sections showing convoluted tubule staining)

• Incubation conditions:

  • Primary antibody: Overnight incubation at 4°C has shown optimal results

  • Secondary detection: Anti-Mouse HRP-DAB systems provide strong visualization

• Detection system selection:

  • Chromogenic detection: HRP-DAB provides good contrast against hematoxylin counterstain

  • Fluorescent detection: Consider using tyramide signal amplification for low-abundance targets

• Negative controls:

  • Isotype-matched irrelevant antibody control

  • Secondary antibody-only control

  • Tissues known to be negative for NAALADL2 expression

How can I resolve inconsistent Western blot results when detecting NAALADL2?

Inconsistent Western blot results with NAALADL2 detection may stem from several factors:

• Sample preparation optimization:

  • Protein extraction method: Use RIPA buffer supplemented with protease inhibitors

  • Sample denaturation: Heat samples at 95°C for 5 minutes in reducing sample buffer

  • Loading amount: Optimize protein loading (typically 20-50 μg total protein)

• Protein transfer considerations:

  • Transfer time and voltage: Extended transfer times (overnight at 30V) may improve transfer of larger NAALADL2 isoforms

  • Membrane selection: PVDF membranes (0.45 μm pore size) typically provide better results than nitrocellulose for this protein

• Antibody selection and dilution:

  • Select antibodies validated specifically for Western blot applications

  • Perform titration experiments to determine optimal concentration

  • Consider using different antibodies targeting distinct epitopes to confirm results

• Protocol modifications for different splice variants:

  • Adjust gel percentage (8-10% polyacrylamide) to better resolve the specific size range

  • Consider gradient gels for simultaneously detecting multiple variants

  • Use specific positive controls for each splice variant

• Signal enhancement strategies:

  • Extended exposure times may be necessary for low-abundance forms

  • Enhanced chemiluminescence substrates with higher sensitivity

  • Consider using fluorescent secondary antibodies for more quantitative analysis

What are the critical parameters for flow cytometry analysis of NAALADL2 expression?

Flow cytometry detection of NAALADL2 requires careful optimization of several parameters:

• Cell preparation protocol:

  • Fixation method: 4% paraformaldehyde (10 minutes)

  • Permeabilization: 0.1% Triton X-100 or commercial permeabilization buffers

  • Cell concentration: 1 × 10^6 cells/mL for optimal staining

• Antibody selection and validation:

  • Use flow cytometry-validated clones (e.g., clone 817225)

  • Include isotype control antibody (e.g., MAB002) at equivalent concentration

  • Use secondary antibodies with appropriate fluorophores (e.g., PE-conjugated anti-mouse IgG)

• Instrument settings optimization:

  • Voltage adjustment based on negative controls

  • Compensation for multiple fluorophores if performing multi-parameter analysis

  • Consistent gating strategy between experiments

• Controls to include:

  • Unstained cells

  • Isotype-matched irrelevant antibody (e.g., MAB002)

  • Single-color controls for compensation

  • Positive control (LNCaP cells have been validated)

• Data analysis considerations:

  • Report data as mean fluorescence intensity (MFI) ratio compared to isotype control

  • Consider frequency of positive cells above threshold based on isotype control

  • Analyze both cell surface and intracellular staining separately if performing dual staining

How can NAALADL2 antibodies be utilized in cancer research beyond their current applications?

NAALADL2 antibodies have significant potential for expanded applications in cancer research:

• Tissue microarray screening:

  • Analyze NAALADL2 expression across multiple cancer types

  • Correlate expression with clinical parameters and patient outcomes

  • Identify new cancer types where NAALADL2 may serve as a biomarker

• Combination with other biomarkers:

  • Co-staining with established cancer markers

  • Development of diagnostic panels including NAALADL2

  • Correlation with genetic alterations in cancer tissues

• Functional studies:

  • Antibody-mediated inhibition of NAALADL2 function

  • Analysis of downstream signaling effects

  • Investigation of enzymatic activity regulation

• Liquid biopsy development:

  • Detection of circulating NAALADL2 in patient serum

  • Correlation with disease status and treatment response

  • Longitudinal monitoring during therapy

• Therapeutic targeting strategies:

  • Development of antibody-drug conjugates

  • Immune cell redirecting therapies

  • Targeted nanoparticle delivery systems

What methodologies are available to investigate the interaction between NAALADL2 and the microRNA regulatory network?

The interaction between NAALADL2 and microRNA networks can be investigated using several sophisticated approaches:

• Crosslinking Immunoprecipitation (CLIP) techniques:

  • HITS-CLIP or PAR-CLIP to identify direct RNA-protein interactions

  • Use NAALADL2 antibodies to pull down associated microRNAs

  • Sequence and analyze captured microRNAs to identify regulatory patterns

• RNA immunoprecipitation (RIP):

  • Use NAALADL2 antibodies to immunoprecipitate the protein

  • Extract and analyze associated RNAs

  • Quantify enrichment of specific microRNAs by qPCR

• Proximity ligation assays (PLA):

  • Visualize and quantify interactions between NAALADL2 and microRNA machinery proteins

  • Provide spatial information about interaction sites within cells

  • Combine with FISH to correlate with RNA distribution

• miRNA mimics and inhibitors:

  • Transfect cells with miR-34a and miR-125a mimics or inhibitors

  • Analyze effects on NAALADL2 expression and function

  • Assess downstream target regulation (e.g., BCL-2)

• CRISPR-based approaches:

  • CRISPR deletion of miRNA binding sites

  • CRISPR activation or interference of NAALADL2 expression

  • Analysis of resulting phenotypic changes

What considerations should be made when developing new NAALADL2 antibodies for emerging research applications?

Development of next-generation NAALADL2 antibodies should consider:

• Epitope selection strategies:

  • Target conserved regions for cross-species applications

  • Target unique regions for splice variant-specific detection

  • Select epitopes not affected by post-translational modifications

• Validation requirements:

  • Knockout/knockdown validation using siRNA or CRISPR

  • Cross-reactivity testing with related proteins

  • Validation across multiple experimental platforms

  • Specificity verification on protein arrays containing target protein plus non-specific proteins

• Application-specific optimization:

  • Super-resolution microscopy compatibility

  • Live-cell imaging suitability

  • Multiplexed imaging compatibility

  • Mass cytometry conjugation potential

• Format considerations:

  • Develop recombinant antibodies for improved reproducibility

  • Create smaller formats (Fab, scFv) for improved tissue penetration

  • Consider conjugation-ready formats with minimal batch variation

• Regulatory compliance:

  • Documentation of validation according to international standards

  • Transparent reporting of validation protocols

  • Detailed information on epitope and specificity