PRDX2 Antibody

What is PRDX2 and why is it important in biological research?

PRDX2 (Peroxiredoxin 2) is a thiol-specific antioxidant enzyme that catalyzes the reduction of hydrogen peroxide and organic hydroperoxides to water and alcohols, respectively. It plays a critical role in:

• Cell protection against oxidative stress by detoxifying peroxides

• Sensing hydrogen peroxide-mediated signaling events

• Regulating intracellular concentrations of H₂O₂ in growth factor and TNF-α signaling cascades

As a key member of the peroxiredoxin family, PRDX2 is one of the most abundant peroxiredoxins and is essential for maintaining cellular redox balance. Its dysregulation has been implicated in various pathological conditions including cancer, neurodegenerative disorders, and cardiovascular diseases, making it a significant target for research .

What applications are PRDX2 antibodies commonly used for in laboratory research?

PRDX2 antibodies are versatile tools used across multiple experimental applications:

These applications have been validated across human, mouse, and rat samples, with specific antibodies showing cross-reactivity between these species .

How does PRDX2 function in the cellular antioxidant defense system?

PRDX2 functions through a cyclical redox mechanism:

• The peroxidatic cysteine residue of PRDX2 reacts with hydrogen peroxide, becoming oxidized to sulfenic acid

• This oxidized cysteine forms a disulfide bond with the resolving cysteine of another PRDX2 molecule

• The resulting PRDX2 dimer is then reduced by thioredoxin, regenerating active PRDX2

This cycle allows PRDX2 to efficiently scavenge ROS and protect cells from oxidative damage. Research demonstrates that PRDX2 plays a particularly important role in vascular smooth muscle cells (VSMCs), where it inhibits hydrogen peroxide production and protects against atherosclerosis progression .

What are the critical factors for selecting the optimal PRDX2 antibody for specific experimental applications?

When selecting a PRDX2 antibody, researchers should consider:

For example, when studying PRDX2 in atherosclerosis models, researchers successfully used antibodies that specifically detect PRDX2 without significant cross-reactivity with other peroxiredoxins to demonstrate its protective role .

What is the optimal protocol for detecting PRDX2 by Western blot?

For optimal PRDX2 detection by Western blot:

• Sample preparation:

  • Extract total protein using RIPA buffer

  • Determine protein concentration using BCA assay

  • Use reducing conditions as PRDX2 forms dimers under oxidizing conditions

• Gel electrophoresis and transfer:

  • Use 12-15% SDS-PAGE gels

  • Transfer to 0.2 μm PVDF membranes

• Antibody incubation:

  • Block with 5% skim milk to prevent non-specific binding

  • Incubate with primary PRDX2 antibody (typically 0.2-10 μg/mL) at 4°C overnight

  • Use appropriate HRP-conjugated secondary antibody

• Detection:

  • Look for a specific band at approximately 24-26 kDa

  • PRDX2 may appear as multiple bands if oxidized forms are present

Studies have demonstrated this protocol effectively detects PRDX2 in various cell lines including Raji human Burkitt's lymphoma cells, A20 mouse B cell lymphoma cells, and NRK rat normal kidney cells .

How can PRDX2 expression be effectively manipulated in experimental models?

To modulate PRDX2 expression in research models:

Overexpression approaches:

• Transfection with pEX4-PRDX2 plasmid has been successfully used in coronary artery vascular smooth muscle cells (CAVSMCs)

• Verify overexpression by qRT-PCR and Western blot

Knockdown approaches:

• siRNA-mediated silencing (siRNA-PRDX2-homo-729) has shown high efficiency

• CRISPR-Cas9 for stable knockout models

In vivo manipulation:

• Xenograft models with sh-PRDX2 have demonstrated reduced tumor growth in colorectal cancer studies

Effective validation of manipulation requires:

• Quantification of PRDX2 mRNA by qRT-PCR

• Protein level confirmation by Western blot

• Functional validation through measurement of H₂O₂ concentration or ROS levels

How should researchers address potential cross-reactivity with other peroxiredoxin family members?

Cross-reactivity with other peroxiredoxin family members is a significant concern:

• Quantify cross-reactivity:

  • Some commercially available antibodies show documented cross-reactivity: 25% with PRDX1 and PRDX4, 10% with PRDX3, and <1% with PRDX5 and PRDX6

  • Test antibodies against recombinant proteins of all PRDX family members

• Validation strategies:

  • Use PRDX2 knockout/knockdown controls to confirm specificity

  • Compare multiple antibodies targeting different epitopes

  • Consider the cellular context - different peroxiredoxins may have distinct subcellular localizations

• Data interpretation:

  • When interpreting Western blot results, be aware that PRDX1 (22 kDa) may be difficult to distinguish from PRDX2 (21.9 kDa)

  • Use densitometry with caution when measuring PRDX2 in samples with high expression of other PRDX family members

If cross-reactivity is unavoidable, complementary approaches like mass spectrometry or specific activity assays may help confirm PRDX2-specific findings.

What are the common challenges in immunohistochemical detection of PRDX2 and how can they be overcome?

Researchers face several challenges when detecting PRDX2 by immunohistochemistry:

In human atherosclerotic tissue samples, researchers successfully overcame these challenges by using antigen retrieval followed by overnight incubation with PRDX2 antibodies, revealing significantly decreased PRDX2 expression in atherosclerotic lesions compared to normal vascular tissue .

How can researchers accurately quantify changes in PRDX2 levels in response to oxidative stress?

For accurate quantification of PRDX2 changes during oxidative stress:

• Consider post-translational modifications:

  • PRDX2 undergoes oxidation during stress, changing its migration pattern

  • Use non-reducing gels to preserve disulfide bonds and distinguish oxidized forms

  • Include controls treated with reducing agents to identify total PRDX2

• Normalize appropriately:

  • Use housekeeping proteins stable under oxidative conditions (β-actin may be affected)

  • Consider total protein normalization methods (Ponceau S, REVERT)

• Complementary approaches:

  • Quantify mRNA levels using qRT-PCR to assess transcriptional changes

  • Measure PRDX2 activity using peroxidase activity assays

  • Monitor subcellular localization changes using immunofluorescence

In studies of TNF-α-induced oxidative stress, researchers successfully tracked PRDX2 function by measuring H₂O₂ concentration and ROS levels in parallel with PRDX2 expression .

How does PRDX2 interact with signaling pathways to regulate cell fate decisions?

PRDX2 interfaces with multiple signaling cascades:

• MAPK Signaling:

  • PRDX2 modulates MAPK pathway activation by regulating H₂O₂ levels

  • In vascular smooth muscle cells, PRDX2 inhibits phenotype alteration and function via MAPK signaling pathway

  • Knockdown of PRDX2 enhances phosphorylation of pathway components

• P38 MAPK/FOXO Pathway:

  • PRDX2 removal inhibits cell cycle progression and autophagy in colorectal cancer cells

  • This regulation occurs through the P38 MAPK/FOXO signaling pathway

• TLR4/NF-κB Signaling:

  • In myocardial tissues, PRDX2 expression changes correlate with alterations in TLR4, p65, and p-p65 levels

  • This suggests PRDX2 may modulate inflammatory responses through NF-κB signaling

These interactions highlight PRDX2's role beyond simple ROS scavenging, positioning it as a key regulator of cell fate through redox-sensitive signaling.

What is the significance of PRDX2 in disease models and how can antibody-based detection inform potential therapeutic strategies?

PRDX2 plays diverse roles across multiple disease models:

Antibody-based detection has revealed these context-dependent roles, suggesting that:

• In cardiovascular diseases, PRDX2 upregulation may be beneficial

• In certain cancers, PRDX2 inhibition could suppress tumor growth

• Monitoring PRDX2 levels might serve as a biomarker for disease progression

These findings highlight the complexity of targeting PRDX2 therapeutically and emphasize the need for context-specific approaches.

How can researchers distinguish between different oxidation states of PRDX2 using antibody-based approaches?

Detecting different PRDX2 oxidation states is methodologically challenging but critical for understanding its function:

• Non-reducing SDS-PAGE:

  • Reduced monomeric PRDX2: ~22 kDa

  • Oxidized dimeric PRDX2: ~44 kDa

  • Hyperoxidized forms: May show altered migration

• Oxidation state-specific antibodies:

  • Some commercial antibodies specifically detect hyperoxidized PRDX2 (Cys-SO₂H/SO₃H)

  • These can be used in parallel with pan-PRDX2 antibodies to determine oxidation ratio

• Technical considerations:

  • Sample preparation must avoid artificial oxidation

  • Use N-ethylmaleimide to alkylate free thiols immediately upon cell lysis

  • Compare samples with and without reducing agents

• Validation approaches:

  • Use H₂O₂ treatment as positive control for oxidation

  • Include PRDX2 mutants (C51S, C172S) as controls for oxidation-resistant forms

When studying oxidative stress in disease models, researchers have successfully combined these approaches to track the functional status of PRDX2, revealing that its protective capacity depends on maintaining its reduced state .

What methodological approaches can resolve contradictory findings regarding PRDX2 function in different experimental systems?

Contradictory findings about PRDX2 function can be resolved through:

• Comprehensive experimental design:

  • Test multiple cell types/tissues simultaneously with identical protocols

  • Include appropriate positive and negative controls

  • Use both gain-of-function and loss-of-function approaches

• Context-dependent analysis:

  • Assess baseline oxidative state of each experimental system

  • Quantify expression levels of other antioxidant enzymes that may compensate for PRDX2

  • Consider tissue/cell-specific interaction partners

• Standardized reporting:

  • Document complete methodological details including:

    • Antibody catalog numbers, dilutions, and incubation conditions

    • Exact composition of buffers and reagents

    • Cell passage numbers and culture conditions

For example, while PRDX2 shows protective effects in cardiovascular models by scavenging ROS and inhibiting VSMC phenotype alteration , it promotes cell proliferation in colorectal cancer models . These seemingly contradictory roles can be reconciled by understanding the distinct redox environments and signaling contexts of each system.