PRDX1 Monoclonal Antibody

What is PRDX1 and what biological functions does it serve?

PRDX1 (Peroxiredoxin 1) is a member of the peroxiredoxin family of antioxidant enzymes that reduce hydrogen peroxide and alkyl hydroperoxides to water and alcohols, respectively. It plays a dual role in cellular physiology: protecting cells against oxidative stress by detoxifying peroxides and functioning as a sensor in hydrogen peroxide-mediated signaling pathways . PRDX1 can also act as a molecular chaperone under conditions of oxidative stress, helping to prevent protein damage .

At the molecular level, PRDX1 contains a conserved N-terminal cysteine (Cys51) that becomes oxidized by H₂O₂ to form cysteine-sulfenic acid. This oxidized form subsequently reacts with the Cys172-SH of another PRDX1 protein, forming a protective disulfide dimer structure . This mechanism is essential for PRDX1's antioxidant functions and its role in redox homeostasis maintenance.

How is PRDX1 involved in immune cell function?

PRDX1 plays differential roles across immune cell populations. Notably, natural killer (NK) cells are more susceptible to oxidative stress compared to T and B cells, partly due to lower endogenous PRDX1 expression . In NK cells, priming with interleukin-15 (IL-15) upregulates PRDX1 expression, which subsequently protects these cells from oxidative stress-induced dysfunction .

The relationship between PRDX1 and oxidative stress susceptibility has significant implications for immunotherapy. Research indicates that PRDX1 overexpression in engineered NK cells, particularly PD-L1-targeting CAR NK cells, confers improved survival and enhanced antitumor function under oxidative stress conditions typical of the tumor microenvironment (TME) . This suggests PRDX1 as a potential target for enhancing cellular immunotherapy approaches against solid tumors.

What applications are PRDX1 monoclonal antibodies suitable for?

Current commercial PRDX1 monoclonal antibodies are validated for multiple research applications:

These antibodies typically recognize a protein with an observed molecular weight of 21-23 kDa on Western blots, which aligns with the predicted molecular weight of PRDX1 (22.1 kDa) . Validation experiments have confirmed specificity in multiple human cell lines, including HepG2 liver cancer cells .

How can PRDX1 expression be accurately quantified in different immune cell subsets?

Quantifying PRDX1 expression in different immune cell populations requires a multi-modal approach. For protein-level assessment, Western blot analysis using validated monoclonal antibodies (0.01-0.03 μg/mL concentration) against PRDX1 is recommended, with β-actin serving as the appropriate loading control .

For gene expression analysis, quantitative PCR (qPCR) has been successfully employed to measure PRDX1 mRNA levels, with normalization to housekeeping genes such as β-actin . When comparing immune cell subsets (T, B, and NK cells), it's critical to isolate pure populations using magnetic or fluorescence-activated cell sorting before analysis.

For single-cell resolution, flow cytometric analysis can be employed using fluorophore-conjugated PRDX1 antibodies, though this requires careful optimization of fixation and permeabilization protocols due to PRDX1's distribution in both cytoplasmic and nuclear compartments. For spatial context in tissues, immunofluorescence microscopy using PRDX1 antibodies (diluted 1:100-200) allows visualization of expression patterns while enabling co-localization studies with cell type-specific markers .

What strategies can overcome oxidative stress-induced loss of NK cell function through PRDX1 modulation?

Several strategies leveraging PRDX1's protective functions can enhance NK cell resilience against oxidative stress:

The most robust evidence supports genetic modification approaches, particularly for cell therapy applications targeting solid tumors where oxidative stress in the tumor microenvironment represents a significant barrier to efficacy .

How does the PRDX1-APE1 interaction impact inflammatory signaling pathways?

The interaction between PRDX1 and APE1 (Apurinic/apyrimidinic endonuclease 1) represents a previously unrecognized regulatory mechanism in inflammatory signaling. PRDX1 physically associates with APE1 under physiological conditions in both nuclear and cytosolic compartments . This interaction has significant functional consequences:

• PRDX1 acts as a negative regulator of APE1's redox activity by blocking its ability to reduce and activate the transcription factor NF-κB .

• When PRDX1 is depleted (via shRNA knockdown), APE1's detection in the nucleus is enhanced, and its redox activity increases .

• The functional consequence of enhanced APE1 redox activity following PRDX1 knockdown is increased expression of the proinflammatory chemokine interleukin-8 (IL-8) .

This regulatory mechanism positions PRDX1 as an anti-inflammatory protein that prevents excessive proinflammatory gene expression by restraining APE1 redox activity. This interaction was confirmed through multiple methodologies, including tandem affinity purification, co-immunoprecipitation, and gel filtration chromatography, which identified a 60 kDa complex containing both APE1 and PRDX1 .

What are the optimal protocols for detecting PRDX1 protein in different subcellular compartments?

Detection of PRDX1 in different subcellular compartments requires tailored approaches:

For Western Blot Analysis of Subcellular Fractions:

• Perform subcellular fractionation to isolate nuclear and cytoplasmic fractions using commercially available kits or differential centrifugation protocols.

• Load 35 μg of protein per lane for reliable detection.

• Use RIPA buffer for protein extraction to ensure efficient solubilization of PRDX1 from different compartments .

• Employ PRDX1 monoclonal antibodies at 0.01-0.03 μg/mL concentration with 1-hour primary antibody incubation .

• Use compartment-specific markers as controls: HDAC1 or Lamin B1 for nuclear fractions and GAPDH or α-tubulin for cytoplasmic fractions.

For Immunofluorescence Microscopy:

• Fix cells with 4% paraformaldehyde (10 minutes at room temperature).

• Permeabilize with 0.1% Triton X-100 (5 minutes).

• Block with 3% BSA in PBS (1 hour).

• Incubate with PRDX1 monoclonal antibody at 1:100-200 dilution (overnight at 4°C) .

• Counterstain with DAPI to visualize nuclei.

• Use confocal microscopy for accurate assessment of nuclear vs. cytoplasmic localization.

When analyzing PRDX1's subcellular distribution, particularly in the context of oxidative stress, it's important to note that rapid changes in localization can occur. Research has demonstrated that PRDX1 detection in the nucleus can be enhanced when its binding partner APE1 is more accessible, as observed in PRDX1 knockdown experiments .

How can researchers effectively validate PRDX1 antibody specificity for their experimental systems?

Comprehensive validation of PRDX1 antibody specificity requires multiple approaches:

• Positive and Negative Controls:

  • Positive: Cell lines known to express PRDX1 (HepG2 is well-validated)

  • Negative: PRDX1 knockdown cells generated via shRNA targeting different regions of the PRDX1 gene (demonstrated to reduce protein levels by ~90%)

• Multiple Detection Methods:

  • Western blot analysis should show a single band at the expected molecular weight (21-23 kDa)

  • Immunoprecipitation followed by mass spectrometry can confirm the identity of the precipitated protein

  • Immunofluorescence patterns should align with known subcellular distribution

• Cross-Reactivity Assessment:

  • Evaluate potential cross-reactivity with other peroxiredoxin family members (PRDX2-6) through recombinant protein panels

  • For antibodies claimed to work across species (human, mouse, rat), confirm specificity in each organism separately

• Validation in Knockout/Knockdown Systems:

  • Utilize CRISPR/Cas9-mediated PRDX1 knockout cells or shRNA knockdown models

  • Successful validation should show absence or significant reduction of signal in these systems

• Epitope Mapping:

  • Confirm that the antibody recognizes the expected epitope (e.g., the region surrounding Arg110 of human PRDX1 for certain commercial antibodies)

When validating monoclonal antibodies against PRDX1, researchers should be aware that some commercial antibodies are produced using specific synthetic peptides (e.g., SDPKRTIAQDYG) corresponding to human PRDX1 , while others use recombinant protein immunogens . This information can be valuable when selecting the most appropriate antibody for a particular application.

What techniques can accurately measure PRDX1 enzymatic activity in experimental samples?

Assessing PRDX1 enzymatic activity requires methodologies that can measure its peroxidase function:

• Hydrogen Peroxide Consumption Assay:

  • Principle: Measures the rate of H₂O₂ decomposition by PRDX1

  • Methodology:

    • Purify PRDX1 from samples via immunoprecipitation

    • Add known concentration of H₂O₂

    • Measure remaining H₂O₂ via colorimetric methods (e.g., FOX assay)

    • Calculate consumption rate compared to controls

• Thioredoxin-Coupled Assay System:

  • Principle: PRDX1 activity depends on the thioredoxin system for recycling

  • Methodology:

    • Combine sample with thioredoxin, thioredoxin reductase, and NADPH

    • Initiate reaction with H₂O₂

    • Monitor NADPH oxidation at 340 nm

    • Activity is proportional to NADPH consumption rate

• Redox Western Blot:

  • Principle: Distinguishes between reduced and oxidized forms of PRDX1

  • Methodology:

    • Treat cells with varying concentrations of oxidants

    • Lyse cells in the presence of alkylating agents to prevent artificial oxidation

    • Perform non-reducing SDS-PAGE to preserve disulfide bonds

    • Probe with PRDX1 antibodies to detect monomeric (reduced) and dimeric (oxidized) forms

• Cellular Hydrogen Peroxide Sensor Systems:

  • Principle: Measures impact of PRDX1 on cellular H₂O₂ levels

  • Methodology:

    • Express genetically-encoded H₂O₂ sensors (e.g., HyPer) in control and PRDX1-modified cells

    • Challenge with oxidative stress

    • Measure fluorescence changes that reflect H₂O₂ levels

    • Compare peroxide elimination rates

When interpreting PRDX1 activity data, researchers should consider that under extreme oxidative conditions, PRDX1 can form high-molecular-weight oligomers that function as molecular chaperones but lose peroxidase activity . This functional switch represents an important consideration when analyzing PRDX1 behavior under different oxidative stress conditions.

How can PRDX1 monoclonal antibodies be utilized to study cancer immunotherapy resistance mechanisms?

PRDX1 monoclonal antibodies provide valuable tools for investigating immunotherapy resistance mechanisms:

• Profiling PRDX1 Expression in Tumor Microenvironments:

  • Immunohistochemistry using PRDX1 antibodies (1:50-300 dilution) can map expression patterns within tumors and the surrounding microenvironment .

  • This allows correlation of PRDX1 levels with oxidative stress markers and treatment response.

  • Multiplex immunofluorescence combining PRDX1 antibodies with immune cell markers can identify which cell populations maintain PRDX1 expression within the tumor.

• Monitoring NK Cell Fitness for Immunotherapy:

  • Western blot analysis with PRDX1 antibodies can assess baseline PRDX1 expression in patient-derived NK cells .

  • This information may help predict NK cell resilience to oxidative stress in the tumor microenvironment.

  • Flow cytometry using permeabilized cells and PRDX1 antibodies can evaluate PRDX1 levels at the single-cell level across immune populations.

• Evaluating Engineered Therapeutic Cells:

  • PRDX1 antibodies enable verification of successful genetic modification in PRDX1-overexpressing CAR NK cells .

  • Immunofluorescence can confirm proper subcellular localization of overexpressed PRDX1.

  • Western blotting quantifies the degree of overexpression relative to endogenous levels.

• Assessing Oxidative Stress Adaptation Mechanisms:

  • PRDX1 antibodies can track changes in expression following exposure to oxidative stress or tumor-derived factors.

  • Co-immunoprecipitation with PRDX1 antibodies followed by mass spectrometry can identify novel interaction partners that emerge under stress conditions.

Research has demonstrated that NK cells are particularly vulnerable to oxidative stress compared to T and B cells, with PRDX1 deficiency identified as a key factor in this susceptibility . This finding has significant implications for NK cell-based cancer immunotherapies, as PRDX1 expression levels may serve as a biomarker for predicting therapeutic efficacy or as a target for enhancing cell therapy products.

What experimental approaches can determine how PRDX1 regulates inflammatory pathways in disease models?

Several experimental approaches using PRDX1 monoclonal antibodies can elucidate its role in inflammatory regulation:

• PRDX1-APE1 Interaction Analysis:

  • Co-immunoprecipitation with PRDX1 antibodies followed by APE1 detection can assess whether this interaction is altered in disease states .

  • Proximity ligation assays can visualize the interaction in situ within tissues.

  • FPLC gel filtration chromatography can isolate the PRDX1-APE1 complex (60 kDa) from cellular extracts under various conditions .

• Functional Impact on NF-κB Signaling:

  • ChIP assays using PRDX1 antibodies can determine whether PRDX1 is present at NF-κB-regulated promoters.

  • Reporter gene assays in PRDX1-manipulated cells can quantify NF-κB transcriptional activity.

  • Immunoblotting for phosphorylated p65 can assess NF-κB activation status in relation to PRDX1 levels.

• Inflammatory Cytokine Production:

  • qPCR and ELISA assays can measure IL-8 production in cells with modulated PRDX1 expression .

  • Multiplex cytokine assays can provide a broader view of the inflammatory landscape affected by PRDX1.

  • Single-cell RNA sequencing of PRDX1-manipulated cells can reveal global transcriptional changes in inflammatory pathways.

• In Vivo Disease Models:

  • Immunohistochemistry with PRDX1 antibodies (1:50-300 dilution) can track expression in tissues during disease progression .

  • Tissue microarrays probed with PRDX1 antibodies can efficiently screen expression across multiple patient samples.

  • Conditional PRDX1 knockout models combined with antibody validation can establish causality in disease pathology.

Research has established that PRDX1 can act as an anti-inflammatory protein by preventing APE1 from activating the transcription factor NF-κB, thereby checking excessive expression of proinflammatory cytokines like IL-8 . This mechanism represents a novel regulatory pathway in inflammation that could have implications for diseases characterized by chronic inflammatory states, including cancer progression where IL-8 plays roles in invasion and metastasis.

How can PRDX1 antibodies be employed to investigate redox-dependent protein-protein interactions?

PRDX1 monoclonal antibodies serve as valuable tools for exploring redox-sensitive protein interactions:

• Redox-Preserved Immunoprecipitation:

  • Methodology:

    • Treat cells with thiol-blocking agents (e.g., MMTS) during lysis to prevent artificial oxidation/reduction of disulfide bonds

    • Perform immunoprecipitation with PRDX1 antibodies

    • Analyze precipitated complexes via western blot or mass spectrometry

  • This approach successfully identified the PRDX1-APE1 complex in nuclear extracts

• Differential Interaction Mapping Under Oxidative Stress:

  • Methodology:

    • Expose cells to graduated levels of H₂O₂ or other oxidants

    • Perform PRDX1 immunoprecipitation at each condition

    • Identify condition-specific interaction partners

    • Validate with reciprocal immunoprecipitation

• In Situ Proximity Ligation Assay:

  • Methodology:

    • Use PRDX1 antibodies in combination with antibodies against suspected interaction partners

    • Apply oligonucleotide-linked secondary antibodies that generate fluorescent signals when in close proximity

    • Quantify interaction signals under different redox conditions

    • This provides spatial context for interactions within cells

• Size Exclusion Chromatography with Antibody Detection:

  • Methodology:

    • Fractionate cellular lysates by gel filtration

    • Analyze fractions by western blot using PRDX1 antibodies

    • Identify high-molecular-weight complexes containing PRDX1

    • Re-probe membranes for potential interaction partners

  • This technique successfully identified a 60 kDa complex containing both PRDX1 and APE1

• Bioluminescence Resonance Energy Transfer (BRET):

  • Methodology:

    • Generate fusion proteins of PRDX1 and potential partners with appropriate BRET donor/acceptor tags

    • Measure interaction dynamics in live cells under various redox conditions

    • Quantify changes in energy transfer as indicator of protein association/dissociation

Research has shown that PRDX1 interactions can be significantly influenced by the cellular redox state. For instance, treatment with (E)-3-[2-(5,6-dimethoxy-3-methyl-1,4-benzoquinonyl)] affects the stability of PRDX1-containing complexes . Additionally, PRDX1's ability to form disulfide-linked dimers and higher-order oligomers under oxidative stress conditions suggests that its interactome likely changes dramatically depending on the redox environment .

What are common challenges when using PRDX1 antibodies and how can they be overcome?

Researchers frequently encounter specific challenges when working with PRDX1 antibodies:

• Multiple Banding Patterns:

  • Challenge: Detection of additional bands beyond the expected 21-23 kDa PRDX1 monomer.

  • Solution:

    • Use reducing agents with caution as PRDX1 forms dimers and oligomers under oxidative stress

    • Include antioxidants in lysis buffers to prevent artificial oxidation

    • Run non-reducing gels in parallel to identify disulfide-linked complexes

    • Confirm band identity through knockdown/knockout controls

• Variable Nuclear vs. Cytoplasmic Detection:

  • Challenge: Inconsistent detection of PRDX1 in nuclear fractions.

  • Solution:

    • Use gentle cell lysis procedures to avoid nuclear leakage

    • Employ thiol-blocking agents during fractionation to preserve protein interactions

    • Consider that PRDX1 nuclear localization may depend on interaction partners like APE1

    • Use properly validated antibodies known to work in immunofluorescence applications

• Cross-Reactivity with Other Peroxiredoxin Family Members:

  • Challenge: Potential recognition of other PRDX isoforms due to sequence similarity.

  • Solution:

    • Select monoclonal antibodies raised against unique PRDX1 epitopes

    • Validate specificity using recombinant PRDX1-6 protein panels

    • Confirm results using genetic knockdown/knockout approaches

    • When possible, use antibodies with validated species cross-reactivity for your model system

• Fixation-Sensitive Epitopes in Immunohistochemistry/Immunofluorescence:

  • Challenge: Loss of antibody reactivity with certain fixation protocols.

  • Solution:

    • Optimize fixation conditions (paraformaldehyde concentration and duration)

    • Test multiple antigen retrieval methods if using FFPE tissues

    • Consider epitope accessibility during permeabilization steps

    • Use properly validated antibodies with published IHC/IF protocols

Researchers should note that several commercial PRDX1 antibodies are available with different properties. Some are produced against synthetic peptides corresponding to specific regions of human PRDX1, while others use recombinant proteins as immunogens . For critical applications, it may be worthwhile to compare multiple antibodies to identify those that perform optimally for the specific experimental system and application.

How can researchers distinguish between different oxidation states of PRDX1 using antibody-based methods?

Distinguishing between PRDX1 oxidation states requires specialized methods:

• Non-Reducing SDS-PAGE Coupled with Western Blotting:

  • Methodology:

    • Prepare lysates in the presence of alkylating agents (e.g., N-ethylmaleimide) to block free thiols

    • Perform SDS-PAGE without reducing agents

    • Blot and probe with PRDX1 antibodies

    • Reduced monomeric PRDX1 appears at ~22 kDa, while disulfide-linked dimers appear at ~44 kDa

• Two-Dimensional Redox Western Blotting:

  • Methodology:

    • Run non-reducing SDS-PAGE in the first dimension

    • Cut the lane and expose to reducing conditions

    • Run the reduced sample in a second dimension SDS-PAGE

    • Blot and probe with PRDX1 antibodies

    • This technique separates different oxidation states into a diagonal pattern

• Modification-Specific Immunoprecipitation:

  • Methodology:

    • Treat cells with dimedone to trap sulfenic acid forms of PRDX1

    • Perform immunoprecipitation with anti-dimedone antibodies

    • Probe western blots with PRDX1 antibodies to quantify sulfenic acid-modified PRDX1

    • Alternatively, immunoprecipitate with PRDX1 antibodies and probe for dimedone adducts

• Conformation-Specific Antibodies:

  • While not mentioned in the search results, developing antibodies specific to particular oxidation states of PRDX1 would be valuable

  • In the interim, researchers can use general approaches to detect PRDX1 hyperoxidation by probing for sulfinic/sulfonic acid forms using anti-PrxSO₂/₃ antibodies

When analyzing PRDX1 oxidation states, it's important to consider that oxidative stress can cause PRDX1 to form high-molecular-weight oligomers that function as molecular chaperones rather than peroxidases . Additionally, under physiological conditions, PRDX1 forms complexes with other proteins such as APE1, which can be preserved using thiol-blocking agents during sample preparation . These interactions may influence the detection and interpretation of different PRDX1 oxidation states.

What emerging research areas could benefit from PRDX1 monoclonal antibodies?

Several frontier research areas could leverage PRDX1 monoclonal antibodies:

• Cancer Immunotherapy Optimization:

  • PRDX1 antibodies could help identify patients likely to benefit from NK cell-based therapies by assessing PRDX1 levels in tumor infiltrating lymphocytes

  • Monitoring PRDX1 expression in therapeutic cell products could serve as a quality control parameter predicting post-infusion efficacy

  • Target validation studies for approaches combining antioxidants with immunotherapy could use PRDX1 antibodies to track cellular redox responses

• Neurodegenerative Disease Research:

  • PRDX1 binds to and regulates JNK and c-Abl kinases, which are implicated in neurodegeneration

  • PRDX1 antibodies could help map expression changes in different neurodegenerative conditions

  • The role of oxidative stress in neurodegeneration could be further elucidated by studying PRDX1-protein interactions in brain tissues

• PTEN-Dependent Cancer Biology:

  • PRDX1 binds to and regulates PTEN phosphatase activity, protecting it from oxidation-induced inactivation

  • PRDX1 antibodies could be used to study this interaction in various cancer models

  • Co-localization studies could reveal spatial regulation of PTEN by PRDX1 in different subcellular compartments

• Redox Signalosome Mapping:

  • Emerging evidence indicates PRDX1 participates in multiple protein-protein interactions influenced by redox state

  • PRDX1 antibodies combined with proximity labeling approaches could identify novel components of redox-regulated signaling hubs

  • Sequential immunoprecipitation with PRDX1 antibodies followed by additional purification steps could isolate intact redox signaling complexes

• Cellular Senescence Mechanisms:

  • Oxidative stress is a key driver of cellular senescence

  • PRDX1 antibodies could track changes in expression and localization during senescence progression

  • Comparisons between senescent and non-senescent cells could reveal altered PRDX1 interactomes

The expanding understanding of PRDX1's multifaceted roles—beyond simple peroxide detoxification to include chaperone functions, protein-protein interactions, and signaling regulation—opens numerous research avenues where specific antibodies will be invaluable tools for mechanistic studies, biomarker development, and therapeutic target validation .