PRDX5 Antibody

What are the key characteristics of PRDX5 and why is it important in cellular research?

PRDX5 belongs to the peroxiredoxin family of antioxidant enzymes that catalyze the reduction of hydrogen peroxide and organic hydroperoxides to water and alcohols. It serves dual functions: detoxifying peroxides and acting as a sensor of hydrogen peroxide-mediated signaling events . PRDX5 is ubiquitously expressed across multiple tissues and cellular compartments, including mitochondria, peroxisomes, and cytoplasm, with tissue-specific expression patterns observed in immunohistochemistry studies .

Research significance stems from PRDX5's involvement in:

• Cellular oxidation state regulation

• Protection against oxidative damage

• Inflammatory processes

• Malignant progression of several cancer types

• Bone development and homeostasis

What applications are PRDX5 antibodies most commonly used for in research?

PRDX5 antibodies have been successfully used in identifying novel protein interactions, such as the binding between PRDX5 and Nrf2 in NSCLC cells under oxidative stress conditions , and between PRDX5 and heterogeneous nuclear ribonucleoprotein K (hnRNPK) during osteoblast differentiation .

How do I select the appropriate PRDX5 antibody for my experimental design?

When selecting a PRDX5 antibody, consider:

• Species reactivity: Most commercial PRDX5 antibodies react with human, mouse, and rat samples . Confirm cross-reactivity if working with other species.

• Antibody type: Both monoclonal and polyclonal options are available. Polyclonal antibodies (like those from rabbit) often provide higher sensitivity but may have more background, while monoclonal antibodies offer higher specificity.

• Validated applications: Ensure the antibody has been validated for your specific application. For example, antibody TA334428 is validated for Western Blot , while 17724-1-AP is validated for WB, IHC, IF, and ELISA applications .

• Target region: Some antibodies target specific regions of PRDX5. For example, PA1838 targets a sequence at the C-terminus of human PRDX5 , while HPA037916 targets a specific immunogen sequence (NKVNLAELFKGKKGVLFGVPGAFTPGCSKTHLPGFVEQAEALKAKGVQVVACLSVNDAFVTGEWGRAHKAEGKV) .

• Isoform recognition: Consider whether the antibody can distinguish between the mitochondrial (isoform L) and peroxisomal/cytoplasmic (isoform S) isoforms of PRDX5 .

What are the recommended storage and handling conditions for PRDX5 antibodies?

For optimal antibody performance and longevity:

• Store at -20°C for long-term storage (typically stable for one year)

• After reconstitution of lyophilized antibodies, store at 4°C for up to one month

• For longer storage of reconstituted antibodies, aliquot and store at -20°C for up to six months

• Avoid repeated freeze-thaw cycles which can degrade antibody quality and performance

• Some antibodies come in stabilized formats (e.g., with 50% glycerol) which helps prevent freeze-thaw damage

• When working with lyophilized formats, reconstitute with the recommended volume of distilled water to achieve optimal concentration (e.g., 0.2ml of distilled water to yield 500μg/ml)

How does PRDX5 interact with Nrf2 in oxidative stress conditions, and what are the implications for cancer research?

The interaction between PRDX5 and Nuclear factor-related factor 2 (Nrf2) represents a significant pathway in cancer development, particularly in NSCLC:

Interaction Mechanism:

• Under H₂O₂ stimulation (oxidative stress conditions), PRDX5 physically interacts with Nrf2 in NSCLC cells

• This interaction was confirmed via immunoprecipitation and immunofluorescence analyses showing partial co-localization in H₂O₂-treated NSCLC cells

• The PRDX5-Nrf2 interaction promotes the expression of NAD(P)H:quinone oxidoreductase 1 (NQO1) protein in NSCLC cells

Clinical Significance:

• High expression of both Nrf2 and PRDX5 is associated with worse prognosis in NSCLC patients

• PRDX5 and Nrf2 expressions are tightly associated with tumor size, clinical TNM stage, lymph node infiltration, differentiation, and Ki-67 expression

• In animal studies, tumors treated with both Nrf2 and PRDX5 shRNA showed significantly reduced growth compared to control groups

Research Applications:

• When studying this interaction, researchers should consider using combined knockdown/knockout approaches targeting both PRDX5 and Nrf2

• Co-immunoprecipitation followed by Western blot is an effective method to detect this interaction

• For subcellular localization studies, dual immunofluorescence staining with confocal microscopy is recommended

What role does PRDX5 play in bone development, and how can PRDX5 antibodies be used to study osteoporosis mechanisms?

PRDX5 has emerging importance in bone biology:

Bone Phenotype in PRDX5 Knockout Models:

• PRDX5 knockout (PRDX5 Ko) male mice exhibit osteoporotic phenotypes

• Micro-CT analysis reveals low bone mineral density (BMD), reduced trabecular number (Tb. N), and increased trabecular bone space (Tb. Sp) in PRDX5 Ko mice compared to wild-type

• PRDX5 Ko mice also show reduced trabecular volume (Tb. V) and thickness (Tb. Th)

Molecular Mechanisms:

• PRDX5 acts as a negative regulator of heterogeneous nuclear ribonucleoprotein K (hnRNPK)-mediated osteocalcin (Bglap) expression

• In PRDX5 Ko mice, RANKL levels are increased by 1.5-fold compared to wild-type, potentially contributing to increased osteoclast activity and bone resorption

• PRDX5 is involved in both osteoclast and osteoblast differentiation processes

Research Applications of PRDX5 Antibodies in Bone Studies:

• Immunohistochemistry using PRDX5 antibodies can help visualize PRDX5 expression in bone tissues

• Western blot analysis can quantify PRDX5 levels in bone-derived cells during differentiation

• Co-immunoprecipitation with PRDX5 antibodies can identify novel binding partners in osteoblasts or osteoclasts

• For studying the function of specific PRDX5 cysteine residues, researchers have used cysteine-to-serine mutants (C48S, C152S) and analyzed their effects on osteoblast differentiation

How can PRDX5 be targeted for cancer therapy, and what methodologies are being developed?

Recent research identifies PRDX5 as a promising therapeutic target, particularly in castration-resistant prostate cancer (CRPC):

Mechanisms in Cancer Progression:

• AR inhibitor treatment in prostate cancer gives rise to a drug-tolerant persister (DTP) state where the thioredoxin/peroxiredoxin pathway, including PRDX5, is upregulated

• PRDX5 promotes AR inhibitor resistance and CRPC development

• In NSCLC, PRDX5 is significantly increased in both specimens and cell lines

• The PRDX5-Nrf2 interaction enhances NQO1 expression and NSCLC progression under oxidative stress

Therapeutic Approaches:

• Inhibition of PRDX5 suppresses DTP cell proliferation in culture

• PRDX5 inhibition dampens CRPC development in animal models

• PRDX5 targeting stabilizes PSA progression and metastatic lesions in patients

• Combined targeting of PRDX5 and Nrf2 shows enhanced anti-tumor effects in NSCLC models

Methodological Considerations for Researchers:

• When developing therapeutic approaches targeting PRDX5, consider:

  • Using PRDX5 antibodies for screening and validation studies to confirm expression in patient samples

  • Employing genetic approaches (siRNA, shRNA, CRISPR) to validate PRDX5 as a target

  • Developing small molecule inhibitors that specifically target PRDX5

  • Assessing effects on oxidative stress markers when targeting PRDX5

What challenges exist in detecting different PRDX5 isoforms, and how can these be addressed methodologically?

Researchers face several challenges when studying PRDX5 isoforms:

Isoform Complexity:

• Alternative transcription start sites result in PRDX5 isoforms targeted to different cellular compartments:

  • Isoform L: targeted to mitochondria

  • Isoform S: targeted to peroxisome/cytoplasm

• These isoforms differ in their N-terminal sequences but share the same functional domains

Molecular Weight Discrepancies:

• Calculated molecular weight of PRDX5 is approximately 22 kDa

• Observed molecular weight varies between sources:

  • 17 kDa according to Proteintech

  • 26 kDa according to Boster Bio

• These differences may result from post-translational modifications, differential processing of isoforms, or variations in gel running conditions

Methodological Solutions:

• Isoform-specific antibodies: Use antibodies that specifically recognize epitopes unique to each isoform

• Subcellular fractionation: Separate mitochondrial, peroxisomal, and cytosolic fractions before Western blot analysis

• Immunofluorescence with organelle markers: Co-stain with mitochondrial or peroxisomal markers to determine localization

• Expression systems with tagged constructs: Generate constructs expressing specific isoforms with different tags for unambiguous identification

• Mass spectrometry: Use proteomics approaches to definitively identify and quantify specific isoforms

How do experimental conditions affect PRDX5 detection, and what optimization strategies are recommended?

Several factors influence the successful detection of PRDX5 using antibodies:

Critical Experimental Variables:

• Sample preparation:

  • Oxidation state of PRDX5 can change during sample preparation, affecting antibody recognition

  • Use of reducing agents in buffers can influence detection of oxidized versus reduced forms

  • Inclusion of protease inhibitors is crucial as PRDX5 can be subject to degradation

• Antigen retrieval for IHC/IF:

  • Recommended methods include TE buffer pH 9.0 or citrate buffer pH 6.0

  • Optimal retrieval conditions may vary depending on fixation method and tissue type

• Blocking conditions:

  • BSA concentration can affect background and specific signal (some antibody formats already contain 0.1% BSA)

  • Alternative blocking agents may be needed for certain applications

Optimization Strategies:

• Western blot optimization:

  • Titrate antibody concentration (recommended range: 1:5000-1:50000)

  • Test different membrane types (PVDF vs. nitrocellulose)

  • Optimize transfer conditions for this relatively small protein

  • Consider gradient gels to better resolve PRDX5 from similar-sized proteins

• IHC optimization:

  • Test multiple dilutions (recommended range: 1:50-1:500)

  • Compare different antigen retrieval methods

  • Include positive control tissues with known PRDX5 expression (e.g., kidney tissue)

  • Consider detection systems with signal amplification for low-expressing samples

• Specialized applications:

  • For detecting PRDX5-protein interactions, optimize immunoprecipitation conditions

  • When studying oxidative stress responses, consider fixation methods that preserve the redox state

  • For isoform-specific detection, combine antibody-based detection with subcellular fractionation

How should experimental design be modified when studying PRDX5 in oxidative stress conditions?

When investigating PRDX5 in the context of oxidative stress:

Sample Handling Considerations:

• Rapid sample processing is crucial to preserve the native oxidation state of PRDX5

• Include N-ethylmaleimide (NEM) in lysis buffers to alkylate free thiols and prevent artificial oxidation during sample preparation

• Use non-reducing conditions in certain experiments to preserve disulfide bonds

Induction of Oxidative Stress:

• H₂O₂ treatment has been successfully used to study PRDX5-Nrf2 interactions (as demonstrated in NSCLC cells)

• Titrate H₂O₂ concentrations carefully (excessive concentrations can cause artificial effects)

• Consider physiologically relevant oxidative stress inducers based on your research context

Detection Methods:

• Use redox Western blotting techniques to distinguish between reduced and oxidized forms of PRDX5

• Consider antibodies that specifically recognize the active site cysteines in different oxidation states

• When studying PRDX5 mutants, remember that Prdx5 forms an intramolecular disulfide bond between Cys48 and Cys152 during its catalytic cycle

What controls and validation steps are essential when working with PRDX5 antibodies?

Rigorous validation ensures reliable results when working with PRDX5 antibodies:

Essential Controls:

• Positive controls:

  • Use tissues/cells known to express PRDX5, such as kidney tissue , HepG2 cells, or U-937 cells

  • Include recombinant PRDX5 protein as a standard for Western blot applications

• Negative controls:

  • PRDX5 knockout or knockdown samples are ideal negative controls

  • For immunohistochemistry, include isotype control antibodies

  • Pre-absorption with immunizing peptide can confirm specificity

• Specificity validation:

  • Test for cross-reactivity with other peroxiredoxin family members

  • Verify antibody specificity across different species if performing comparative studies

  • Confirm signal corresponds to predicted molecular weight (with consideration for isoform variations)

Validation Approaches:

• Genetic validation:

  • siRNA or shRNA-mediated knockdown of PRDX5 should reduce antibody signal

  • CRISPR/Cas9 knockout provides definitive validation

  • Overexpression systems can confirm antibody detection sensitivity

• Multi-method concordance:

  • Compare results across different detection methods (WB, IHC, IF)

  • Use multiple antibodies targeting different epitopes of PRDX5

  • Correlate protein detection with mRNA expression data

How can PRDX5 antibodies be effectively used to study protein-protein interactions in disease models?

PRDX5 has important interaction partners that mediate its biological functions:

Key Interaction Partners:

• Nrf2 in NSCLC and oxidative stress response

• hnRNPK in osteoblast differentiation

• Other potential interactors in specific disease contexts

Methodological Approaches:

• Co-immunoprecipitation (Co-IP):

  • Use PRDX5 antibodies to pull down PRDX5 and associated proteins

  • Verify interactions by immunoblotting for suspected binding partners

  • Consider reverse Co-IP (pull down with partner antibody, detect PRDX5)

  • Remember that interactions may be condition-dependent (e.g., H₂O₂-stimulated interaction with Nrf2)

• Proximity Ligation Assay (PLA):

  • Allows visualization of protein-protein interactions in situ

  • Requires antibodies from different species against each interaction partner

  • Provides spatial information about where interactions occur within cells

• Immunofluorescence co-localization:

  • Has been successfully used to demonstrate partial co-localization of PRDX5 and Nrf2 in H₂O₂-treated NSCLC cells

  • Requires high-quality antibodies suitable for immunofluorescence

  • Confocal microscopy provides better resolution for co-localization studies

• Bimolecular Fluorescence Complementation (BiFC):

  • Engineer fusion constructs of PRDX5 and suspected partners with split fluorescent protein fragments

  • Interaction brings fragments together, generating fluorescence

  • Allows visualization of interactions in living cells

What are the special considerations when using PRDX5 antibodies in clinical or translational research?

When applying PRDX5 antibody detection to clinical specimens or translational studies:

Sample Considerations:

• Fixation methods significantly impact epitope preservation and antibody access

• For FFPE samples, optimize antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0 recommended)

• Consider tissue-specific expression patterns of PRDX5 when interpreting results

• Remember that PRDX5 expression may vary with disease state, treatment, or oxidative stress conditions

Clinical Correlations:

• PRDX5 and Nrf2 expressions are associated with tumor size, clinical TNM stage, lymph node infiltration, differentiation, and Ki-67 expression in NSCLC

• High expression of Nrf2 and PRDX5 correlates with worse prognosis in NSCLC patients

• PRDX5 inhibition stabilizes PSA progression and metastatic lesions in prostate cancer patients

Standardization Approaches:

• Use tissue microarrays (TMAs) to enable consistent processing and comparison across multiple samples

• Include reference standards when performing quantitative analyses

• Consider automated staining platforms to reduce technical variability

• Develop scoring systems that account for both staining intensity and distribution

What emerging technologies and approaches are enhancing PRDX5 research?

The field of PRDX5 research continues to evolve with new methodologies:

Advanced Technologies:

• CRISPR/Cas9 gene editing:

  • Enables precise manipulation of PRDX5 gene

  • Can be used to generate knockout models or introduce specific mutations

  • Allows study of cysteine residue functions by creating site-specific mutations

• Single-cell analysis techniques:

  • Single-cell RNA-seq can reveal cell-type specific expression patterns of PRDX5

  • Mass cytometry (CyTOF) with PRDX5 antibodies enables high-dimensional analysis

  • Spatial transcriptomics provides information about PRDX5 expression in tissue context

• Redox proteomics:

  • Enables system-wide analysis of changes in protein oxidation states

  • Can identify PRDX5 substrates and interaction partners under oxidative stress

  • Provides insight into the broader redox network in which PRDX5 functions

• In vivo imaging:

  • Development of PRDX5 activity biosensors

  • Non-invasive monitoring of redox status in animal models

  • Correlation of PRDX5 activity with disease progression or treatment response

Therapeutic Development:

• Small molecule inhibitors targeting PRDX5 show promise for cancer therapy

• Peptide-based approaches targeting specific PRDX5 interactions

• Combined targeting strategies (e.g., PRDX5 and Nrf2 for NSCLC)

• Nanoparticle delivery systems for PRDX5-targeting agents