Customize NUDT16 Antibody

Description

Definition and Target Specificity

The NUDT16 antibody is a specialized immunological tool designed to detect and study the NUDT16 protein, a member of the Nudix hydrolase family. This enzyme is critical for RNA decapping, nucleotide metabolism, and DNA repair processes . The antibody binds specifically to endogenous NUDT16, enabling its visualization and quantification in experimental settings.

Biological Functions of NUDT16

NUDT16 is a multifunctional enzyme with distinct roles:

RNA Decapping: Cleaves m⁷G/m²²⁷G caps from snoRNAs and mRNAs, leaving a 5'-monophosphate .
Nucleotide Sanitization: Hydrolyzes non-canonical purines (e.g., inosine diphosphate (IDP), deoxyinosine diphosphate (dIDP)) to prevent their incorporation into DNA/RNA .
DNA Repair: Regulates homologous recombination (HR) and alternative end-joining (Alt-EJ) by stabilizing CtIP and modulating PARylation .

Substrate Specificity:

Substrate	Activity (kₐₜ/Kₘ)	Role in Cellular Processes
IDP/dIDP	Highest	Prevents DNA/RNA damage
GDP/dGDP	Moderate	Maintains nucleotide pool integrity
m⁷G-capped RNA	Metal-dependent	snoRNA/mRNA degradation

A. Western Blot (WB)

Detects endogenous NUDT16 at ~21 kDa in lysates (e.g., mouse small intestine) .
Validated in knockdown studies: siRNA-mediated NUDT16 silencing reduces protein levels, confirming antibody specificity .

B. Immunohistochemistry (IHC)

Localizes NUDT16 predominantly in nucleoli and nucleoplasm .
Used in human kidney tissue studies with antigen retrieval protocols .

C. Functional Studies

DNA Repair Mechanisms:
- NUDT16 deficiency increases CtIP ADP-ribosylation, leading to ubiquitination and degradation .
- Silencing NUDT16 reduces HR and Alt-EJ repair efficiency .
Cell Cycle Regulation:
- Knockdown causes S-phase arrest and ssDNA accumulation .

A. Validation Methods

siRNA Knockdown: Reduced NUDT16 levels correlate with increased inosine in RNA and ssDNA breaks .
Co-Immunoprecipitation (Co-IP): Confirmed interactions with CtIP and PARP1 .
Proximity Ligation Assay (PLA): Visualized NUDT16-CtIP complexes in U2OS cells .

Disease Implications

Huntington’s Disease (HD):
- Expanded CAG RNAs silence NUDT16, causing DNA damage and apoptosis .
- Small-molecule inhibitors (e.g., DB213) restore NUDT16 expression, rescuing DNA repair defects .

Product Specs

Buffer

Preservative: 0.03% Proclin 300
Composition: 50% Glycerol, 0.01M PBS, pH 7.4

Form

Liquid

Lead Time

Made-to-order (14-16 weeks)

Synonyms

NUDT16 antibody; NUDX16 antibody; At3g12600 antibody; T2E22.9 antibody; Nudix hydrolase 16 antibody; mitochondrial antibody; AtNUDT16 antibody; EC 3.6.1.- antibody

Target Names

NUDT16

Uniprot No.

Q9LHK1

Target Background

Function

NUDT16 antibody is believed to facilitate the hydrolysis of certain nucleoside diphosphate derivatives.

Database Links

KEGG: ath:AT3G12600

STRING: 3702.AT3G12600.1

UniGene: At.20911

Protein Families

Nudix hydrolase family

Subcellular Location

Mitochondrion.

Tissue Specificity

Expressed in roots, leaves, stems and inflorescences.

Q&A

Basic Research Questions

How to validate NUDT16 antibody specificity in human cell lines?

Methodology:
- Perform siRNA-mediated knockdown of NUDT16 and compare protein levels via Western blot (WB) using the antibody. A ≥70% reduction in signal confirms specificity .
- Use CRISPR/Cas9 knockout (KO) cell lines (e.g., MCF10A or U2OS) as negative controls .
- Cross-validate with mass spectrometry after immunoprecipitation (IP) to confirm target identity .

Validation Step	Expected Outcome	Source Example
Knockdown/KO	Reduced band intensity
IP-MS	Identification of NUDT16 peptides

What are the optimal conditions for detecting NUDT16 in immunofluorescence (IF)?

Protocol:
- Fixation: 4% paraformaldehyde (15 min).
- Permeabilization: 0.1% Triton X-100 (10 min).
- Primary antibody dilution: 1:200–1:800 .
- Critical controls: Include KO cells and secondary antibody-only samples to rule out non-specific binding.

How to address non-specific bands in Western blot?

Solutions:
- Pre-clear lysates with Protein A/G beads before IP.
- Use high-stringency wash buffers (e.g., 500 mM NaCl, 0.1% SDS).
- Validate with multiple cell lines (e.g., HeLa, K-562) to confirm consistent banding patterns .

Advanced Research Questions

How to design experiments investigating NUDT16’s role in PARylation-dependent DNA repair?

Experimental Framework:
- Induce double-strand breaks (DSBs) with ionizing radiation (IR) or CRISPR/Cas9.
- Assess CtIP-NUDT16 interaction via proximity ligation assay (PLA) or co-IP under PARP inhibition (e.g., Olaparib) .
- Quantify DNA resection using DIvA system qPCR or RPA2 phosphorylation (RPA2pS4/S8 foci) .

Key Assay	Readout	Functional Insight
PLA	NUDT16-CtIP foci	Interaction dynamics
DIvA-qPCR	ssDNA %	Resection efficiency

How to resolve contradictions in NUDT16’s enzymatic activity across studies?

Analytical Approach:
- Compare substrate specificity under varying cofactors (Mn²⁺ vs. Mg²⁺). For example, NUDT16 exhibits broader activity with Mn²⁺ .
- Use in vitro ADP-ribosylation assays with purified proteins to isolate enzymatic activity from cellular modifiers .
- Validate findings in isogenic cell lines (WT vs. catalytic mutants like H24A/R50AD52A) .

What controls are critical for studying NUDT16-CtIP interactions in PARylation contexts?

Essential Controls:
- PARP inhibitor treatment (e.g., Olaparib) to block ADP-ribosylation .
- CtIP mutants (e.g., RSS3A) lacking ADP-ribosylation sites .
- PLA with single-antibody controls to exclude false positives .

Control Type	Purpose	Example Result
PARPi	Disrupt PARylation-dependent binding	Reduced PLA foci
CtIP mutant	Test site-specific interaction	Fewer PLA dots

How to optimize ChIP-seq for studying NUDT16’s chromatin recruitment?

Optimization Steps:
- Crosslink cells with 1% formaldehyde (10 min).
- Fragment chromatin to 200–500 bp via sonication.
- Include IgG and no-antibody controls to assess background.
- Validate targets via qPCR at known binding loci (e.g., U8 snoRNA regions) .

Data Interpretation Guidelines

For Conflicting Results:
- Compare cell-type-specific roles (e.g., NUDT16 promotes Alt-EJ in U2OS but not in EJ7-GFP cells) .
- Evaluate antibody lot variability using independent validation data (e.g., vs. ).

Conflict Source	Resolution Strategy
Cell-line variability	Use multiple models (e.g., MCF10A, U2OS)
Antibody specificity	Validate with KO lines in parallel

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NUDT16 Antibody