NQO2 Antibody

What is NQO2 and what are its primary cellular functions?

NQO2 is a 231 amino acid protein that functions as a flavoenzyme catalyzing the reduction of quinones to hydroquinones. Unlike its isozyme NQO1, NQO2 uniquely utilizes dihydronicotinamide riboside (NRH) as a cofactor instead of NAD(P)H. This enzyme plays a crucial role in cellular defense mechanisms by detoxifying potentially harmful compounds, thereby protecting cells from oxidative stress, cytotoxicity, and mutagenicity. NQO2 contains a specific metal binding site and several regulatory elements in its promoter region that are essential for its expression and function .

The cellular functions of NQO2 extend beyond detoxification to include:

• Protection against oxidative damage

• Modulation of cellular redox status

• Involvement in protein stabilization pathways

• Contribution to hematopoietic regulation

NQO2 is widely expressed across various tissues, with particularly high expression in the liver and testis. Its expression can be induced by environmental toxins such as TCDD, suggesting its role in xenobiotic responses .

How can researchers distinguish between NQO1 and NQO2 in experimental systems?

While NQO1 and NQO2 are isozymes with similar catalytic functions, they can be distinguished experimentally through several approaches:

For definitive identification, researchers should employ specific antibodies that do not cross-react between these isozymes. The NQO2 Antibody (A-5) has been validated for detecting NQO2 protein of mouse, rat, and human origin without cross-reactivity to NQO1 .

What experimental techniques are most effective for studying NQO2 expression?

Several experimental techniques can be effectively employed to study NQO2 expression:

• Western Blotting: Using specific antibodies like NQO2 Antibody (A-5) allows for quantitative assessment of protein expression levels. This technique can detect NQO2 from multiple species including human, mouse, and rat samples .

• Immunofluorescence: For studying cellular localization and expression patterns of NQO2 in intact cells or tissues. Conjugated antibodies (FITC, PE, or Alexa Fluor) can enhance detection sensitivity .

• ELISA: Provides quantitative measurement of NQO2 in biological samples with high sensitivity.

• qRT-PCR: For measuring NQO2 mRNA expression levels to understand transcriptional regulation.

• Immunohistochemistry: For analyzing tissue-specific expression patterns and localization of NQO2.

When analyzing NQO2 expression, researchers should consider using multiple complementary techniques to confirm findings, especially in new experimental systems or when studying novel conditions affecting NQO2 regulation.

What are the optimal protocols for using NQO2 antibodies in Western blotting?

For optimal Western blotting results with NQO2 antibodies, researchers should follow these methodological considerations:

Sample Preparation:

• Use RIPA or NP-40 based lysis buffers containing protease inhibitors

• Include antioxidants (e.g., DTT or β-mercaptoethanol) in sample buffers to prevent oxidation

• Heat samples at 95°C for 5 minutes in Laemmli buffer before loading

Gel Electrophoresis and Transfer:

• 12-15% SDS-PAGE gels are recommended for optimal resolution of NQO2 (~26 kDa)

• Use PVDF membranes for better protein retention and signal-to-noise ratio

• Transfer at 100V for 1 hour or 30V overnight at 4°C

Antibody Incubation:

• Block membranes with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Dilute primary NQO2 antibody (e.g., NQO2 Antibody A-5) at 1:500-1:1000 in blocking buffer

• Incubate overnight at 4°C with gentle agitation

• Wash 3-4 times with TBST, 5-10 minutes each

• Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

• Perform enhanced chemiluminescence detection

Critical Controls:

• Include positive control (liver or testis lysate)

• Include negative control (tissues with low NQO2 expression)

• Consider using NQO2 knockout/knockdown samples as specificity controls

Researchers should optimize antibody concentrations for their specific experimental system, as optimal dilutions may vary depending on the sample type and detection method used.

How can NQO2 antibodies be effectively used in immunoprecipitation studies?

Immunoprecipitation (IP) using NQO2 antibodies allows researchers to investigate protein-protein interactions involving NQO2. The following methodological approach is recommended:

Sample Preparation:

• Lyse cells in non-denaturing buffer (e.g., 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.5) with protease inhibitors

• Clear lysate by centrifugation (14,000 × g for 10 minutes at 4°C)

• Pre-clear with Protein A/G beads to reduce non-specific binding

Immunoprecipitation:

• Add NQO2 antibody (2-5 μg) to 500-1000 μg of protein lysate

• Incubate overnight at 4°C with gentle rotation

• Add Protein A/G beads and incubate for 2-4 hours at 4°C

• Wash beads 4-5 times with lysis buffer

• Elute proteins by boiling in Laemmli buffer

• Analyze by SDS-PAGE and Western blotting

For Co-Immunoprecipitation Studies:

• Perform IP with NQO2 antibody

• After elution, probe Western blots with antibodies against potential interacting proteins (e.g., C/EBPα or 20S proteasome components)

• Validate interactions with reverse co-IP where appropriate

When studying NQO2's role in protein stabilization, researchers can specifically investigate its interaction with C/EBPα, as NQO2 has been shown to stabilize C/EBPα against 20S proteasomal degradation .

For agarose-conjugated antibodies (such as NQO2 Antibody A-5 AC), the protocol can be modified by directly adding the conjugated antibody to the lysate, eliminating the need for separate Protein A/G beads .

What approaches can be used to measure NQO2 enzymatic activity in experimental samples?

Measuring NQO2 enzymatic activity is crucial for understanding its functional role in various biological contexts. Researchers can employ several methodological approaches:

Spectrophotometric Assays:

• Basic Principle: Monitor the reduction of electron acceptors (e.g., DCPIP, MTT, or cytochrome c) in the presence of NQO2 and its specific cofactor NRH

• Protocol Outline:

  • Prepare reaction mixture containing buffer, electron acceptor, and enzyme sample

  • Initiate reaction by adding NRH

  • Monitor absorbance changes at appropriate wavelength (e.g., 600 nm for DCPIP)

  • Calculate activity based on extinction coefficient

Substrate-Specific Activity Assays:

• Menadione Reduction: NQO2 catalyzes the reduction of menadione (Km = 4.3 ± 0.1 μM) much more efficiently than acetaminophen (Km = 417 ± 10 μM)

• Acetaminophen as Substrate: To assess NQO2's interaction with acetaminophen, researchers can measure its weak substrate activity in a specialized assay system

In-Cell Activity Measurements:

• ROS Detection: Since NQO2 can generate reactive oxygen species, particularly superoxide anions, fluorescent probes like MitoSOX can be used to indirectly assess NQO2 activity

• Cellular Thermal Shift Assay (CETSA): This technique can be used to monitor NQO2 engagement with substrates or inhibitors in live cells

Inhibitor Studies:

• Include quercetin as a positive control inhibitor of NQO2

• Compare activity in the presence and absence of specific inhibitors

• Use resveratrol as another known NQO2 inhibitor for validation

When measuring NQO2 activity, it's essential to include appropriate controls to distinguish NQO2-specific activity from other quinone reductases, particularly NQO1. This can be achieved by using NQO2-specific inhibitors or comparing activity in NQO2 knockout/knockdown models .

How can researchers investigate NQO2's role in oxidative stress responses?

NQO2's involvement in oxidative stress responses can be investigated through several experimental approaches:

ROS Detection Assays:

• Superoxide Detection: Use MitoSOX fluorescence probe, which is particularly sensitive to superoxide anions. This approach has been validated to demonstrate NQO2-dependent superoxide production in response to compounds like acetaminophen .

• Comparative ROS Analysis: Compare ROS levels between:

  • Wild-type cells vs. NQO2 knockdown/knockout cells

  • Cells treated with NQO2 inhibitors vs. controls

  • Cells overexpressing NQO2 vs. controls

Glutathione Status Assessment:

• Measure reduced (GSH) and oxidized (GSSG) glutathione levels to evaluate cellular redox status

• Correlate GSH/GSSG ratios with NQO2 expression levels

• Analyze GSH depletion kinetics in response to oxidative challenges in cells with varying NQO2 expression

Protein Oxidation Analysis:

• Use NQO2 antibodies in conjunction with antibodies against oxidative stress markers

• Perform dual immunofluorescence to correlate NQO2 expression with protein carbonylation or nitrosylation

• Assess lipid peroxidation products in relation to NQO2 activity

Functional Studies:

• Measure cell viability under oxidative stress conditions in relation to NQO2 expression

• Assess mitochondrial function (membrane potential, ATP production) in cells with modified NQO2 levels

• Evaluate Ca²⁺ homeostasis using fluorescent Ca²⁺ indicators like Fluo-3, as NQO2 has been shown to modulate Ca²⁺ levels in response to compounds that induce oxidative stress

When investigating NQO2's role in oxidative stress, researchers should consider using multiple, complementary approaches to build a comprehensive understanding of its function in specific cellular contexts.

What is the relationship between NQO2 and protein stability, and how can it be studied?

NQO2 plays a significant role in protein stability, particularly through protection against 20S proteasomal degradation. This function can be studied using the following approaches:

Protein Stability Assays:

• Cycloheximide Chase: Treat cells with cycloheximide to inhibit protein synthesis, then monitor degradation rates of target proteins (e.g., C/EBPα) in the presence or absence of NQO2

• Pulse-Chase Analysis: Use radioisotope or biotin labeling to track protein turnover rates

20S Proteasome Interaction Studies:

• In Vitro Degradation Assays:

  • Incubate in vitro-translated proteins (e.g., C/EBPα) with purified 20S proteasome

  • Assess degradation in the presence or absence of recombinant NQO2

  • Monitor protein levels over time by Western blotting

• Co-Immunoprecipitation:

  • Use NQO2 antibodies to pull down protein complexes

  • Probe for 20S proteasome subunits (e.g., α5 subunit) or target proteins like C/EBPα

Genetic Manipulation Approaches:

• Expression Studies:

  • Overexpress NQO2 and monitor levels of target proteins

  • Use siRNA to knockdown NQO2 and assess effects on protein stability

  • Research has shown that overexpression of NQO2 in HL-60 cells leads to up-regulation of C/EBPα and PU.1, while siRNA-mediated inhibition of NQO2 in U937 cells leads to down-regulation of both proteins

• Domain Mapping:

  • Create NQO2 mutants to identify regions required for protein stabilization

  • Perform competition assays with peptides to disrupt specific interactions

Proteasome Inhibitor Studies:

• Compare effects of 20S-specific versus general proteasome inhibitors

• Assess whether NQO2-dependent protein stabilization occurs independent of ubiquitination

• Use MG132 as a control proteasome inhibitor

This research area is particularly important as NQO2 stabilization of C/EBPα protects against myeloproliferative disease, highlighting NQO2's role beyond simple detoxification functions .

How does NQO2 interact with acetaminophen, and what implications does this have for toxicology research?

NQO2 has been identified as a reactive oxygen species-generating off-target for acetaminophen, with significant implications for understanding drug toxicity mechanisms. This interaction can be studied through several approaches:

Binding Studies:

• Thermal Shift Assays:

  • In vitro thermal shift assays show that acetaminophen binds to and stabilizes NQO2

  • Cellular thermal shift assays (CETSA) confirm binding in live cells

  • ITDRF-CETSA shows that approximately 1 mM extracellular acetaminophen concentration is required to observe binding under experimental conditions

• Comparative Binding Analysis:

  • Acetaminophen binds NQO2 with lower affinity compared to known inhibitors like resveratrol

  • Structurally similar compounds like AMAP (acetaminophen analog) also bind NQO2

Enzymatic Activity Assessment:

• Substrate Activity:

  • Acetaminophen acts as a weak NQO2 substrate (Km = 417 ± 10 μM) compared to menadione (Km = 4.3 ± 0.1 μM)

  • Activity is abrogated by quercetin, indicating NQO2-specific effects

  • No substrate activity is observed with NQO1, confirming specificity

• ROS Production:

  • Strong correlation (R² = 0.499) exists between NQO2 substrate activity in vitro and superoxide production in cells

  • Acetaminophen-induced MitoSOX signal (indicating superoxide) is reversed by superoxide scavenger MnTBAP

  • ROS formation occurs independently of NAPQI (N-acetyl-p-benzoquinone imine) formation and P450 activity

Toxicological Implications:

• Tissue Specificity:

  • NQO2 is highly expressed in liver and kidney, the main sites of acetaminophen toxicity

  • This suggests NQO2-mediated ROS production may contribute to organ-specific damage

• Calcium Homeostasis:

  • NQO2 modulates acetaminophen-induced changes in Ca²⁺ levels

  • Inhibition of NQO2 reduces Ca²⁺ level changes caused by acetaminophen

• Protective Strategies:

  • NQO2 inhibitors (resveratrol and quercetin) have been shown to alleviate acetaminophen toxicity in mice

  • This suggests potential therapeutic approaches targeting NQO2 for acetaminophen overdose

This research area represents an important intersection between drug metabolism, oxidative stress, and cellular toxicity pathways. Future studies in animal models, particularly NQO2 knockout mice, could provide definitive validation of NQO2's role in acetaminophen toxicity .

How does NQO2 function in hematopoietic regulation and myeloid development?

NQO2 plays a significant role in hematopoietic regulation, particularly in myeloid development and protection against myeloproliferative disorders. This function can be studied through several experimental approaches:

Genetic Model Systems:

• NQO2 Knockout Studies:

  • NQO2⁻/⁻ mice exhibit myeloid hyperplasia and significantly increased granulocyte levels in peripheral blood

  • These mice show increased sensitivity to γ radiation-induced myeloproliferative disease and B cell lymphomas

  • These phenotypes suggest NQO2's protective role against hematological disorders

Transcription Factor Regulation:

• C/EBPα and PU.1 Relationship:

  • NQO2 stabilizes C/EBPα against 20S proteasomal degradation

  • C/EBPα is a key transcription factor found predominantly in immature myeloid cells

  • C/EBPα regulates PU.1 gene expression, which is expressed in both lymphoid and myeloid cells

  • Deregulation of C/EBPα is associated with myeloid transformation

• Experimental Evidence:

  • Overexpression of NQO2 in HL-60 cells leads to up-regulation of both C/EBPα and PU.1

  • siRNA-mediated inhibition of NQO2 in U937 cells leads to down-regulation of both C/EBPα and PU.1

  • These results demonstrate that NQO2 controls C/EBPα stability, which subsequently affects PU.1 expression

Proteasomal Degradation Protection:

• In Vitro Degradation Assays:

  • In vitro-translated C/EBPα incubated with purified 20S proteasome shows significant degradation within 1 hour

  • This degradation can be studied in the context of NQO2's protective effects

Methodological Approaches:

• Cell Line Models:

  • Myeloid cell lines (HL-60, U937) can be used to study NQO2's role in hematopoietic regulation

  • These models allow for genetic manipulation (overexpression, knockdown) to assess NQO2's effects

• Protein Analysis Techniques:

  • Western blotting using specific antibodies against NQO2, C/EBPα, and PU.1

  • Immunoprecipitation to study protein-protein interactions

  • 20S proteasome activity assays to assess degradation protection

Understanding NQO2's role in hematopoietic regulation has significant implications for research into myeloproliferative disorders and potential therapeutic approaches targeting this pathway.

What experimental systems are most effective for studying NQO2's role in hematological disorders?

To effectively study NQO2's role in hematological disorders, researchers can employ several experimental systems:

In Vitro Cell Models:

• Myeloid Cell Lines:

  • HL-60 and U937 cells have been successfully used to study NQO2's effects on C/EBPα and PU.1 regulation

  • These cell lines allow for genetic manipulation (overexpression, siRNA knockdown) and biochemical analyses

• Primary Hematopoietic Cells:

  • Bone marrow-derived cells from wild-type and NQO2 knockout mice

  • Human CD34+ hematopoietic stem and progenitor cells with NQO2 modulation

In Vivo Models:

• NQO2 Knockout Mice:

  • NQO2⁻/⁻ mice show myeloid hyperplasia and increased granulocyte levels

  • These mice demonstrate increased sensitivity to radiation-induced myeloproliferative disease

  • Bone marrow transplantation studies can determine cell-autonomous versus non-cell-autonomous effects

• Radiation-Induced Myeloproliferative Disease Models:

  • Comparing wild-type and NQO2⁻/⁻ mice following radiation exposure

  • Analyzing bone marrow and peripheral blood for myeloid abnormalities

  • Studying the development of B cell lymphomas in relation to NQO2 status

Molecular and Biochemical Approaches:

• Protein Stability Analysis:

  • Cycloheximide chase experiments to assess C/EBPα stability

  • In vitro 20S proteasomal degradation assays with recombinant proteins

  • Co-immunoprecipitation studies to identify protein interactions

• Transcription Factor Activity Assays:

  • Luciferase reporter assays for C/EBPα and PU.1 activity

  • ChIP assays to assess transcription factor binding to target genes

  • Gene expression analysis of myeloid differentiation markers

Flow Cytometry Applications:

• Differentiation Analysis:

  • Multi-parameter flow cytometry to assess myeloid differentiation markers

  • Cell cycle analysis to determine proliferation status

  • Apoptosis assays to evaluate cell survival

• Functional Assays:

  • Colony-forming unit assays to assess progenitor cell function

  • Cell differentiation assays using myeloid-inducing agents

  • ROS detection to correlate oxidative stress with differentiation status

When studying NQO2's role in hematological disorders, it's important to combine multiple experimental approaches to build a comprehensive understanding of its functions. Researchers should validate findings across different experimental systems and consider both cell-autonomous and systemic effects of NQO2 modulation.

What are common challenges when using NQO2 antibodies, and how can they be addressed?

Researchers may encounter several challenges when working with NQO2 antibodies. Here are common issues and their solutions:

Non-specific Binding:

• Problem: Multiple bands or high background in Western blots

• Solutions:

  • Optimize blocking conditions (try 5% BSA instead of milk for phosphorylation studies)

  • Increase washing stringency (add 0.1% SDS or increase salt concentration in wash buffer)

  • Use more specific antibody clones (A-5 monoclonal shows high specificity)

  • Pre-absorb antibody with non-specific proteins

  • Include NQO2 knockout/knockdown controls to identify specific bands

Weak Signal:

• Problem: Low detection of NQO2 despite known expression

• Solutions:

  • Optimize antibody concentration (titrate between 1:100-1:1000)

  • Increase protein loading (up to 50 μg per lane)

  • Use enhanced sensitivity detection systems (ECL-Plus or similar)

  • Try conjugated antibody versions (HRP-conjugated NQO2 antibody)

  • Extend primary antibody incubation (overnight at 4°C)

  • Consider antigen retrieval methods for immunohistochemistry

Cross-reactivity with NQO1:

• Problem: Inability to distinguish between NQO1 and NQO2

• Solutions:

  • Use validated antibodies with confirmed specificity for NQO2

  • Include NQO1 knockout controls

  • Perform parallel experiments with NQO1-specific antibodies

  • Verify with functional assays using specific substrates or inhibitors

Immunoprecipitation Difficulties:

• Problem: Poor yield in IP experiments

• Solutions:

  • Use agarose-conjugated NQO2 antibodies (SC-271665 AC)

  • Optimize lysis conditions (try different detergents: NP-40, CHAPS)

  • Increase antibody amount (3-5 μg per mg of protein lysate)

  • Extend incubation time (overnight at 4°C with gentle rotation)

  • Pre-clear lysates thoroughly to reduce background

  • Use protein A/G beads appropriate for the antibody species and isotype

Fixation Sensitivity in Immunofluorescence:

• Problem: Loss of epitope recognition after fixation

• Solutions:

  • Compare multiple fixation methods (4% PFA, methanol, acetone)

  • Reduce fixation time (10-15 minutes may be sufficient)

  • Include permeabilization step (0.1-0.5% Triton X-100)

  • Try antigen retrieval methods (heat-induced or enzymatic)

  • Use fluorophore-conjugated primary antibodies (FITC, PE, or Alexa Fluor conjugates)

By systematically addressing these challenges, researchers can optimize NQO2 antibody performance across various applications and experimental systems.

How should researchers validate NQO2 antibody specificity in their experimental systems?

Proper validation of NQO2 antibody specificity is crucial for generating reliable research data. Researchers should implement the following comprehensive validation strategies:

Genetic Validation Approaches:

• Knockout/Knockdown Controls:

  • Compare antibody reactivity in wild-type versus NQO2 knockout/knockdown samples

  • Use siRNA-mediated knockdown of NQO2 (as demonstrated in U937 cells)

  • For transient validation, use at least two different siRNA sequences targeting NQO2 to control for off-target effects

• Overexpression Controls:

  • Express tagged versions of NQO2 (FLAG-tagged, V5-tagged) and confirm detection

  • Compare endogenous versus overexpressed protein patterns

  • HL-60 cells transfected with FLAG-NQO2 plasmid can serve as positive controls

Biochemical Validation Methods:

• Mass Spectrometry Confirmation:

  • Immunoprecipitate NQO2 and analyze by mass spectrometry

  • Compare detected peptides with known NQO2 sequence

• Peptide Competition Assays:

  • Pre-incubate antibody with immunizing peptide before application

  • Specific binding should be blocked by relevant peptides

• Molecular Weight Verification:

  • Confirm detection at the expected molecular weight (~26 kDa for NQO2)

  • Validate with recombinant protein standards

Multi-Antibody Approach:

• Epitope Diversity:

  • Use antibodies recognizing different epitopes of NQO2

  • Compare reactivity patterns across techniques

  • For example, combine monoclonal (A-5) with other available antibodies

• Concordance Analysis:

  • Results should be consistent across different antibodies targeting NQO2

  • Discrepancies may indicate non-specific binding

Functional Validation:

• Activity Correlation:

  • Correlate antibody detection with NQO2 enzymatic activity

  • Compare NQO2 inhibition (by quercetin or other inhibitors) with protein detection

• Cellular Thermal Shift Assay (CETSA):

  • Verify antibody detection of thermally stabilized NQO2 following ligand binding

  • This approach confirms both antibody specificity and functional NQO2 detection

Cross-Species Validation:

• Species Comparison:

  • Test antibody against NQO2 from multiple species (human, mouse, rat)

  • Confirm expected cross-reactivity or species specificity

• Tissue Expression Patterns:

  • Verify detection in tissues known to express high levels of NQO2 (liver, kidney)

  • Compare with published expression patterns

Thorough validation ensures that experimental findings reflect true NQO2 biology rather than artifacts of non-specific antibody interactions. Researchers should document validation steps and include appropriate controls in all experimental reports.