NT5C2 Antibody, HRP conjugated

What is NT5C2 and what is its biological significance?

NT5C2 (also known as Cytosolic purine 5'-nucleotidase) is a broad specificity enzyme that catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates. The enzyme plays a critical role in regulating purine nucleoside/nucleotide pools within the cell, which is essential for numerous cellular processes . Additionally, NT5C2 possesses phosphotransferase activity, enabling it to transfer phosphate from a donor nucleoside monophosphate to an acceptor nucleoside, preferably inosine, deoxyinosine, and guanosine . The enzyme shows highest activities for IMP and GMP followed by dIMP, dGMP, and XMP, though it can also catalyze the transfer of phosphates from pyrimidine monophosphates with lower efficiency .

What are the known alternative names and identifiers for NT5C2?

NT5C2 is referenced in scientific literature under multiple designations, which is important to recognize when conducting comprehensive literature searches. These alternative names include NT5B, NT5CP, PNT5, Cytosolic 5'-nucleotidase II, Cytosolic IMP/GMP-specific 5'-nucleotidase, Cytosolic nucleoside phosphotransferase 5'N, High Km 5'-nucleotidase, and cN-II . The protein is cataloged in UniProt with the identifier P49902 .

Why is NT5C2 significant in cancer research, particularly leukemia?

NT5C2 has emerged as a critical protein in cancer research due to its association with chemotherapy resistance. Mutations in NT5C2 have been identified in relapsed pediatric B lymphoblastic leukemia patients and are associated with resistance to purine analogue therapies such as 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) . These mutations confer increased enzymatic activity to NT5C2, resulting in enhanced clearance of thiopurine metabolites and consequently reduced treatment efficacy . Notably, patients harboring NT5C2 mutations tend to relapse early, within 36 months of initial diagnosis, indicating the clinical relevance of NT5C2 status in treatment outcome prediction .

What are the technical specifications of commercially available NT5C2 antibodies?

Several NT5C2 antibodies are available commercially with varying specifications. The HRP-conjugated polyclonal antibody (e.g., catalog #A48537) is generated in rabbits using recombinant Human Cytosolic purine 5'-nucleotidase protein (amino acids 487-553) as the immunogen . This antibody is supplied in liquid form with a buffer composition of 0.03% Proclin 300 as a preservative and 50% Glycerol in 0.01M PBS at pH 7.4 . Other available formats include non-conjugated antibodies like ab96084, which is generated against a recombinant fragment within Human NT5C2 (amino acids 100-350) . These antibodies typically undergo Protein G purification with purity exceeding 95% .

What experimental applications are NT5C2 antibodies validated for?

The applications for NT5C2 antibodies vary by product. The HRP-conjugated antibody is primarily validated for ELISA applications . Other NT5C2 antibodies, such as ab96084, are suitable for Western blotting (WB) and immunohistochemistry on paraffin-embedded sections (IHC-P) . When conducting novel applications or working with different species, researchers should consider validation status. Some antibody combinations have been fully tested and are covered by manufacturer guarantees, while others may be predicted to work based on sequence homology but lack experimental validation .

What is the proper storage and handling protocol for NT5C2 Antibody, HRP conjugated?

To maintain antibody integrity and activity, NT5C2 Antibody, HRP conjugated should be stored at -20°C or -80°C upon receipt . It is essential to avoid repeated freeze-thaw cycles as this can degrade the antibody and the conjugated HRP enzyme . For working solutions, aliquoting is recommended to minimize freeze-thaw events. The antibody is supplied in a stabilizing buffer containing glycerol, which helps maintain activity during storage, but proper temperature control remains critical for preserving the HRP conjugate functionality.

What is the recommended protocol for using NT5C2 Antibody in Western blotting?

When using NT5C2 antibodies for Western blotting, the following methodology is recommended based on published research: After standard SDS-PAGE separation and membrane transfer, block the membrane using an appropriate blocking solution. Incubate with the primary NT5C2 antibody at a 1:5000 dilution, followed by washing and secondary antibody application with a horseradish peroxidase-conjugated anti-species antibody . For HRP-conjugated primary antibodies, this secondary incubation step can be omitted. Detection should be performed using enhanced chemiluminescence reagents. Important controls should include a β-actin loading control, which can be detected using a horseradish peroxidase-conjugated β-actin antibody (such as clone 8H10D10, #12262 from Cell Signaling) .

How can NT5C2 antibodies be utilized to investigate NT5C2 mutations in leukemia research?

Investigating NT5C2 mutations in leukemia research requires a multifaceted approach. While antibodies are valuable for protein detection and quantification, they typically cannot distinguish between wild-type and mutant forms without mutation-specific antibodies. For comprehensive mutation analysis, researchers should consider:

This integrated approach provides more comprehensive insights than antibody detection alone.

What experimental designs effectively measure NT5C2 activity in relation to drug resistance?

To assess the relationship between NT5C2 activity and drug resistance, researchers have implemented several experimental designs:

• Cell viability assays: Comparing survival of cells expressing wild-type versus mutant NT5C2 (or alternatively spliced variants) when treated with increasing concentrations of purine analogues such as 6-MP .

• Metabolite analysis: Measuring intracellular accumulation of thiopurine nucleotides following 6-MP treatment in cells with different NT5C2 variants to correlate enzymatic activity with drug metabolism .

• In vivo xenograft models: Implanting luciferase-expressing leukemia cells with different NT5C2 variants into immunodeficient mice and tracking treatment response through bioluminescence imaging .

• Cross-sensitivity testing: Evaluating whether NT5C2 mutations that confer resistance to thiopurines also affect sensitivity to other drugs, such as the increased sensitivity to mizoribine observed in cells with hyperactive NT5C2 .

These complementary approaches provide robust evidence of NT5C2's role in drug resistance mechanisms.

How do mutations and alternative splicing in NT5C2 affect enzyme function and drug resistance?

NT5C2 mutations identified in relapsed leukemia patients, particularly the hotspot mutation R238W, confer gain-of-function properties to the enzyme . Similarly, an alternatively spliced isoform of NT5C2 that includes cryptic exon 6a (NT5C2 ex6a) creates a proteoform with enhanced enzymatic activity comparable to the R238W mutation . Both genetic alterations result in:

• Increased nucleotidase activity, enhancing the dephosphorylation of active thiopurine metabolites

• Reduced intracellular accumulation of thioguanoside nucleotides following 6-MP treatment

• Comparable levels of resistance to 6-MP treatment in cell-based assays

• Similar growth retardation phenotypes in vitro and in vivo, attributed to depletion of intracellular nucleotide pools

• Increased sensitivity to mizoribine, an inosine monophosphate dehydrogenase (IMPDH) inhibitor

These findings suggest that both genetic mutations and alternative splicing can create convergent resistance mechanisms through similar biochemical alterations to NT5C2 function.

What are the potential therapeutic implications of NT5C2 research?

Research on NT5C2 has revealed several potential therapeutic avenues:

• NT5C2 inhibitors: Development of small-molecule inhibitors such as CRCD2 could potentially overcome resistance mediated by hyperactive NT5C2 .

• Alternative drug selection: Patients with NT5C2 mutations or NT5C2 ex6a expression might benefit from treatments that do not rely on the same metabolic pathways affected by NT5C2 hyperactivity.

• Collateral sensitivity exploitation: The heightened sensitivity to mizoribine exhibited by cells with hyperactive NT5C2 suggests that this FDA-approved drug could be repurposed for treating resistant leukemias .

• Diagnostic applications: Detection of NT5C2 mutations or alternatively spliced variants could serve as biomarkers for predicting treatment response and guiding therapy selection.

• Combination therapies: Understanding the metabolic consequences of NT5C2 hyperactivity could inform rational design of drug combinations that exploit the altered nucleotide metabolism in resistant cells.

These approaches represent promising directions for translating NT5C2 research into clinical applications.

How does the presence of NT5C2 mutations correlate with clinical outcomes in leukemia?

Clinical data indicates that NT5C2 mutations are significantly associated with early relapse in acute lymphoblastic leukemia patients. Specifically, patients harboring NT5C2 mutations tend to relapse within 36 months of initial diagnosis (p=0.03) . This correlation suggests that NT5C2 mutation status could serve as a prognostic marker for identifying patients at higher risk of early treatment failure. Interestingly, in at least one documented case, a patient's leukemia cells simultaneously displayed both the hotspot mutation R367Q and the alternatively spliced NT5C2 ex6a isoform, raising questions about tumor heterogeneity and possible cooperation between genetic and transcriptional mechanisms of resistance . The clinical significance of such dual alterations remains an important area for further investigation.

What are common technical challenges when using NT5C2 antibodies and how can they be resolved?

When working with NT5C2 antibodies, researchers may encounter several technical challenges:

For HRP-conjugated antibodies specifically, additional considerations include protecting the reagent from light and avoiding exposure to heavy metals or sodium azide, which can inhibit peroxidase activity.

What controls are essential when studying NT5C2 in experimental systems?

Robust experimental design for NT5C2 studies should include the following controls:

• Positive controls: Cell lines or samples with confirmed NT5C2 expression

• Negative controls: Samples lacking NT5C2 expression or where expression has been knocked down

• Loading controls: β-actin or other housekeeping proteins for normalization in Western blotting

• Isotype controls: For immunohistochemistry or flow cytometry applications

• Expression controls: When studying mutant forms or splice variants, parallel analysis of wild-type NT5C2 is essential for comparative assessment

• Functional controls: For drug resistance studies, include both sensitive and known resistant cell lines to benchmark the effects of NT5C2 variants

These controls ensure reliable interpretation of results and facilitate troubleshooting when unexpected outcomes occur.