NUDT26 Antibody

What is NUDT26 and what is its biological function in plants?

NUDT26 belongs to the Nudix hydrolase family proteins in Arabidopsis thaliana. While specific functions of NUDT26 are still being investigated, Nudix family proteins generally function as "housekeeping" enzymes that hydrolyze various nucleoside diphosphate derivatives. By comparison with related proteins like NUDT2, which catalyzes the asymmetric hydrolysis of diadenosine tetraphosphate to yield AMP and ATP, NUDT26 likely performs similar nucleotide metabolism functions within plant cells . Some Nudix hydrolases also exhibit RNA decapping activity, suggesting potential roles in RNA metabolism and gene expression regulation. The protein is identified in UniProt database as Q9CAF2 .

What are the key specifications of commercially available NUDT26 antibodies?

The commercially available NUDT26 antibody (e.g., CSB-PA866413XA01DOA) is a rabbit polyclonal antibody produced against recombinant Arabidopsis thaliana NUDT26 protein. The antibody is purified using antigen affinity methods and supplied in liquid form containing PBS pH 7.4 with 50% glycerol and 0.03% Proclin 300 as preservative . It is an IgG isotype antibody that has been validated for specific applications including ELISA and Western blotting for antigen identification. The antibody is specific to Arabidopsis thaliana and is available as a made-to-order product with lead times of approximately 14-16 weeks .

What applications is the NUDT26 antibody validated for?

Current validation confirms the NUDT26 antibody's utility in ELISA and Western blot (WB) applications for antigen identification . Unlike some other antibodies like NUDT2 which have been validated for immunofluorescence and immunochromatography , NUDT26 antibody validation appears more limited to these specific applications. When designing experiments, researchers should consider performing their own validation if using the antibody for additional applications beyond those already confirmed.

What is the recommended protocol for Western blot analysis using NUDT26 antibody?

For Western blot applications with NUDT26 antibody, researchers should follow a standard protocol with the following specific considerations:

• Sample preparation: Extract total protein from Arabidopsis thaliana tissue under conditions that preserve protein integrity.

• Protein separation: Load approximately 90 μg of total protein extract per lane (based on comparable antibody protocols for plant proteins) .

• Transfer: Use standard PVDF or nitrocellulose membrane.

• Blocking: Block with 5% non-fat milk or BSA in TBST.

• Primary antibody: Dilute NUDT26 antibody at 1:1000 in blocking solution (extrapolated from similar antibody protocols) .

• Secondary antibody: Use anti-rabbit IgG conjugated with HRP.

• Detection: Employ enhanced chemiluminescence (ECL) detection.

When running controls, consider including samples from wild-type and NUDT26 overexpression lines to confirm specificity, similar to approaches used with other plant protein antibodies .

How should NUDT26 antibody be stored and handled to maintain reactivity?

The NUDT26 antibody should be stored at -20°C or -80°C upon receipt . The lyophilized form offers greater stability for long-term storage. After reconstitution, it's advisable to make small aliquots to avoid repeated freeze-thaw cycles which can degrade antibody quality . Before opening tubes containing lyophilized antibody, briefly spin them to ensure no material is lost from the cap or sides. For working solutions, maintain the antibody on ice during experiment setup and avoid extended periods at room temperature .

What controls should be included when working with NUDT26 antibody?

Recommended controls include:

• Positive control: Tissue samples known to express NUDT26, such as Arabidopsis thaliana wild-type seedlings under conditions that induce expression.

• Negative control: Tissues from knockout or knockdown NUDT26 mutant lines of Arabidopsis thaliana.

• Loading control: Probe for constitutively expressed proteins such as actin or tubulin.

• Secondary antibody control: Omit primary antibody to assess non-specific binding of the secondary antibody.

This approach parallels control strategies employed with other plant protein antibodies, such as those demonstrated for AHB1 antibody testing .

How can researchers validate the specificity of NUDT26 antibody in experimental settings?

To validate antibody specificity, researchers should implement multiple strategies:

• Genetic validation: Compare signal between wild-type plants and NUDT26 knockout/knockdown mutants.

• Recombinant protein validation: Use purified recombinant NUDT26 protein as a positive control.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before application to confirm that specific binding is blocked.

• Signal correlation: Correlate protein levels detected by the antibody with mRNA expression levels from qPCR.

• Mass spectrometry validation: Confirm the identity of the immunoprecipitated protein band through mass spectrometry analysis.

These approaches align with best practices in antibody validation for research applications .

How can NUDT26 antibody be used to study protein-protein interactions?

For protein interaction studies, researchers can adapt the following methodologies:

• Co-immunoprecipitation (Co-IP): Use NUDT26 antibody to pull down NUDT26 and associated proteins from plant extracts, then analyze by mass spectrometry.

• Proximity ligation assay (PLA): Combine NUDT26 antibody with antibodies against suspected interaction partners to visualize protein proximity in situ.

• FRET-based approaches: Use NUDT26 antibody labeled with a fluorophore alongside antibodies against potential interaction partners to assess energy transfer.

These approaches should be optimized specifically for plant tissues, considering the cell wall and other plant-specific structural features that may impact antibody accessibility.

What are the potential cross-reactivities with other members of the Nudix family?

• Perform Western blot analysis with recombinant proteins of related Nudix family members.

• Include samples from plants overexpressing various Nudix proteins as controls.

• Consider using knockout lines of multiple Nudix family members to confirm signal specificity.

What are common issues encountered with NUDT26 antibody in Western blot applications and how can they be resolved?

Common issues and their solutions include:

This troubleshooting approach draws on established practices for plant protein antibody applications .

How can researchers optimize NUDT26 antibody dilution for different applications?

Optimization should follow a systematic approach:

• For Western blot: Start with 1:1000 dilution as a baseline , then test a dilution series (1:500, 1:1000, 1:2000) to determine optimal signal-to-noise ratio.

• For ELISA: Begin with 1:1000 dilution and adjust based on signal strength.

• For immunofluorescence (if attempting): Start with more concentrated dilutions (1:100 to 1:500) as plant tissues often require higher antibody concentrations.

Document results quantitatively using signal-to-noise measurements to determine optimal conditions for each specific application.

What are the best extraction methods to preserve NUDT26 protein integrity for antibody detection?

For optimal protein extraction from Arabidopsis tissues:

• Use buffer containing: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, with freshly added protease inhibitors.

• Grind tissue thoroughly in liquid nitrogen before adding extraction buffer.

• Maintain cold temperature throughout extraction (0-4°C).

• Centrifuge at high speed (14,000 × g, 15 min, 4°C) to remove cell debris.

• Assess protein concentration using Bradford assay or BCA method.

• Add reducing agent (β-mercaptoethanol or DTT) to sample buffer just before loading.

This methodology aligns with successful approaches for other plant proteins .

How can NUDT26 antibody contribute to understanding plant stress responses?

NUDT26 antibody can be valuable for investigating plant stress responses through:

• Protein expression analysis: Monitor NUDT26 protein levels under various stress conditions (drought, salt, temperature, pathogen exposure) using Western blot.

• Subcellular localization studies: Determine if NUDT26 protein relocalization occurs during stress using immunofluorescence microscopy.

• Post-translational modification analysis: Investigate whether stress induces modifications of NUDT26 through combining the antibody with phospho-specific or other PTM detection methods.

• Comparisons with other Nudix family members: Study potential compensatory expression changes among different Nudix proteins during stress responses.

These approaches parallel methods used in studying other plant proteins like non-symbiotic hemoglobin (AHB1), which shows expression changes under submergence stress .

What emerging methodologies can incorporate NUDT26 antibody in plant research?

Emerging methodologies that could benefit from NUDT26 antibody include:

• Single-cell proteomics: Adaptation of NUDT26 antibody for use in highly sensitive single-cell protein detection methods.

• Microfluidic antibody arrays: Integration into high-throughput screening platforms to assess NUDT26 expression across multiple conditions simultaneously.

• CRISPR-based tagging combined with antibody detection: Using CRISPR to add tags to endogenous NUDT26 that can enhance antibody detection sensitivity.

• Computational antibody design approaches: Similar to those used for therapeutic antibodies , computational methods could potentially enhance NUDT26 antibody specificity and affinity through targeted amino acid substitutions.

How does NUDT26 research compare with investigations of other Nudix family proteins like NUDT2?

While NUDT26 is studied primarily in plant systems (Arabidopsis thaliana), NUDT2 has been extensively studied in mammalian systems including human, mouse, and rat models . Comparative research opportunities include:

• Functional conservation: Investigating whether NUDT26 performs similar nucleotide hydrolysis functions as mammalian NUDT2, which catalyzes diadenosine tetraphosphate hydrolysis .

• Structural analysis: Comparing structural features of plant and mammalian Nudix proteins using antibodies for immunoprecipitation followed by structural studies.

• Evolutionary biology: Using antibodies to trace the expression and function of Nudix proteins across diverse plant species, drawing parallels to the evolution of these proteins in animals.

• Methodological adaptations: Applying techniques developed for NUDT2 antibody applications in mammalian systems to enhance NUDT26 antibody applications in plant research.

This comparative approach provides valuable evolutionary insights into conserved nucleotide metabolism mechanisms across diverse organisms.