PRDX3 Antibody

What is the recommended protocol for using PRDX3 antibody in Western blotting?

For optimal Western blotting results with PRDX3 antibody, follow these methodological steps:

• Sample preparation: Lyse cells or tissues in RIPA buffer containing protease inhibitors. Keep samples on ice throughout processing to prevent protein degradation.

• Protein quantification: Determine protein concentration using BCA or Bradford assay to ensure equal loading.

• Gel electrophoresis: Load 20-30 μg of protein per lane on a 12% SDS-PAGE gel.

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane at 100V for 1 hour.

• Blocking: Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute PRDX3 antibody at 1:5000-1:50000 in blocking solution and incubate overnight at 4°C.

• Washing: Wash membrane 3 times with TBST, 5 minutes each.

• Secondary antibody: Incubate with HRP-conjugated anti-rabbit secondary antibody for 1 hour.

• Detection: Develop using ECL substrate and capture images.

The expected band for PRDX3 should appear at 26-28 kDa, consistent with its calculated molecular weight of 27 kDa . For validation, positive controls should include lysates from HeLa cells, HEK-293 cells, MCF-7 cells, or LNCaP cells, which have been confirmed to express PRDX3 .

How should PRDX3 antibody be optimized for immunohistochemistry staining?

For optimal immunohistochemistry results with PRDX3 antibody, implement this methodology:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) sections cut at 4-5 μm thickness.

• Deparaffinization and rehydration: Process sections through xylene and graded alcohols to water.

• Antigen retrieval: This is a critical step for PRDX3 detection. Use TE buffer pH 9.0 for optimal results, though citrate buffer pH 6.0 may serve as an alternative .

• Endogenous peroxidase blocking: Incubate sections with 3% hydrogen peroxide for 10 minutes.

• Protein blocking: Apply 5% normal goat serum for 30 minutes.

• Primary antibody: Dilute PRDX3 antibody at 1:50-1:500 and incubate overnight at 4°C .

• Detection system: Apply a polymer detection system compatible with rabbit primary antibodies.

• Chromogen development: Develop with DAB substrate.

• Counterstain: Apply hematoxylin, dehydrate, and mount.

For validation, human lung cancer tissue has been confirmed to show positive staining for PRDX3 . When evaluating staining, note that PRDX3 primarily localizes to mitochondria, resulting in a granular cytoplasmic pattern. Studies have shown increased PRDX3 expression in ovarian cancer tissues compared to normal ovarian tissues, with medium staining observed in 8 out of 9 ovarian cancer tissues examined .

What controls are essential when using PRDX3 antibody in immunofluorescence experiments?

When conducting immunofluorescence experiments with PRDX3 antibody, include these essential controls:

• Positive control: HepG2 cells have been confirmed to express PRDX3 and should be used as a positive control .

• Negative control: Omit primary antibody while including all other steps to assess non-specific binding of the secondary antibody.

• Blocking peptide control: Pre-incubate the PRDX3 antibody with its immunizing peptide before application to confirm specificity.

• Mitochondrial co-localization control: Since PRDX3 is primarily localized to mitochondria, co-stain with a mitochondrial marker such as MitoTracker or anti-COX IV antibody .

• Knockdown control: If possible, include cells where PRDX3 has been knocked down using siRNA or shRNA to confirm antibody specificity .

For optimal staining, dilute PRDX3 antibody at 1:200-1:800 in antibody diluent and incubate overnight at 4°C . Expect to observe a distinctive mitochondrial staining pattern, which has been confirmed by cell fractionation and confocal microscopy analyses . When interpreting results, be aware that some studies have shown that a fraction of PRDX3 may also localize to the cell membrane, particularly in prostate cancer cell lines where this localization can be androgen-regulated .

How can weak or absent PRDX3 signal in Western blot be troubleshooted?

When encountering weak or absent PRDX3 signal in Western blot, systematically address these potential issues:

• Protein extraction optimization:

  • Ensure complete lysis using appropriate buffer containing detergents like NP-40 or Triton X-100

  • Include protease inhibitors to prevent degradation

  • Maintain cold temperature throughout processing

• Antibody dilution adjustment:

  • PRDX3 antibody working range is 1:5000-1:50000; try a more concentrated dilution if signal is weak

  • Prepare fresh antibody dilution

  • Consider longer incubation times (overnight at 4°C)

• Sample processing considerations:

  • Add reducing agent (β-mercaptoethanol) to sample buffer

  • Avoid excessive heating which may cause protein aggregation

  • Process samples immediately after collection

• Detection system optimization:

  • Use a more sensitive detection reagent (enhanced ECL)

  • Increase exposure time when imaging

  • Consider using more sensitive membrane (PVDF)

• Positive control verification:

  • Include lysates from HeLa, HEK-293, MCF-7, or LNCaP cells as positive controls

  • The expected molecular weight for PRDX3 is 26-28 kDa

If signal remains problematic after these adjustments, consider validating with alternative PRDX3 antibodies or confirming target expression at the mRNA level. Also note that PRDX3 expression varies by tissue type, with brain, heart, and various cancer tissues typically showing stronger expression .

How can PRDX3 antibody be utilized to investigate mitochondrial reactive oxygen species regulation in immunological research?

PRDX3 antibody can be instrumental in investigating mROS regulation in immunological research through these methodological approaches:

• Isolation and analysis of mitochondrial fractions:

  • Harvest cells and isolate mitochondria using differential centrifugation

  • Verify mitochondrial fraction purity using markers such as COX IV

  • Assess PRDX3 levels in mitochondrial fractions by Western blot (1:5000-1:50000 dilution)

  • Compare PRDX3 levels across different immune cell types and activation states

• Dual fluorescence microscopy for localization and functional studies:

  • Co-stain cells with PRDX3 antibody (1:200-1:800) and MitoSOX Red (mROS indicator)

  • Analyze correlation between PRDX3 expression and mROS levels

  • Quantify fluorescence intensity using image analysis software

• PRDX3 knockdown studies for functional assessment:

  • Generate PRDX3 knockdown cells using shRNA or siRNA

  • Verify knockdown efficiency by Western blot

  • Measure mROS levels using flow cytometry with MitoSOX Red

  • Compare mROS levels between control and PRDX3 knockdown cells under basal and stimulated conditions

• Bacterial infection assays to assess immunological function:

  • Infect control and PRDX3 knockdown cells with bacteria (e.g., Salmonella Typhimurium)

  • Assess bacterial survival and killing efficiency

  • Correlate with mROS levels and PRDX3 expression

Research has demonstrated that PRDX3 knockdown THP-1 cells exhibit significantly higher mROS levels compared to control cells, both at baseline and in response to lipopolysaccharide (LPS) stimulation . Furthermore, PRDX3 knockdown cells show enhanced resistance to Salmonella Typhimurium infection, indicating that PRDX3 is functionally important in bactericidal activity through the regulation of mROS . These findings suggest PRDX3 antibody can be valuable in studying the role of mitochondrial redox regulation in immune responses to bacterial pathogens.

What methodological approaches are recommended for investigating PRDX3's role in cancer drug resistance?

Investigating PRDX3's role in cancer drug resistance requires careful methodological planning:

• Baseline expression analysis across sensitive and resistant cell lines:

  • Compare PRDX3 expression between drug-sensitive and drug-resistant cancer cell lines

  • Use Western blot with PRDX3 antibody (1:5000-1:50000) for protein quantification

  • Perform RT-qPCR to determine if regulation occurs at mRNA or protein level

  • Compare results across multiple cell line models

• Patient sample analysis for clinical correlation:

  • Stain tissue microarrays with PRDX3 antibody (1:50-1:500)

  • Categorize staining as negative, low, medium, or high

  • Correlate expression levels with clinical treatment response data

  • Compare PRDX3 levels between treatment-naïve and post-treatment samples

• PRDX3 expression manipulation studies:

  • Generate PRDX3 knockdown models using shRNA or CRISPR-Cas9

  • Create PRDX3 overexpression models by transfection

  • Verify expression changes by Western blot

  • Test drug sensitivity in modified cells compared to controls

• Oxidative stress and apoptosis assessment:

  • Measure ROS levels using fluorescent probes

  • Assess apoptotic response to therapeutic agents

  • Evaluate activation of pro-apoptotic pathways in relation to PRDX3 levels

Studies have shown that PRDX3 expression is associated with platinum resistance in ovarian cancer, with significantly higher expression in platinum-resistant serous ovarian cancer compared to platinum-sensitive counterparts . Research in prostate cancer has revealed that PRDX3 is upregulated in antiandrogen-resistant LNCaP cell lines at the protein level but not RNA level, suggesting post-transcriptional regulation . These resistant cells show upregulation of the tricarboxylic acid (TCA) pathway and resistance to H₂O₂-induced apoptosis, which can be restored by PRDX3 knockdown . These findings indicate PRDX3 is a potential therapeutic target in treatment-resistant cancers.

How should experiments be designed to differentiate between mitochondrial and membrane-associated PRDX3?

Designing experiments to differentiate between mitochondrial and membrane-associated PRDX3 requires specialized approaches:

• Subcellular fractionation with Western blot analysis:

  • Perform differential centrifugation to isolate mitochondrial, cytosolic, and membrane fractions

  • Use ultracentrifugation with density gradients for high-purity membrane isolation

  • Analyze PRDX3 distribution by Western blot (1:5000-1:50000 dilution)

  • Include markers for each fraction: COX IV (mitochondria), Na⁺/K⁺ ATPase (plasma membrane), GAPDH (cytosol)

• Confocal microscopy with co-localization analysis:

  • Perform immunofluorescence staining with PRDX3 antibody (1:200-1:800)

  • Co-stain with MitoTracker for mitochondria

  • Co-stain with membrane markers (e.g., wheat germ agglutinin)

  • Analyze co-localization using Pearson's correlation coefficient

  • Use z-stack imaging to confirm true co-localization

• Cell surface protein biotinylation:

  • Biotinylate cell surface proteins using non-permeable biotin reagents

  • Isolate biotinylated proteins with streptavidin beads

  • Probe for PRDX3 in both biotinylated (membrane) and non-biotinylated fractions

  • Include controls: cytosolic protein (negative) and known membrane protein (positive)

• Hormone regulation studies (for prostate cancer cells):

  • Treat cells with androgen (e.g., dihydrotestosterone) or anti-androgens

  • Analyze changes in membrane-associated PRDX3 versus mitochondrial PRDX3

  • Compare protein levels in different fractions by Western blot

Research has demonstrated that while the majority of PRDX3 is localized to mitochondria, there is evidence for androgen-regulated PRDX3 at the cell membrane in prostate cancer cells . These findings suggest distinct functional roles for PRDX3 based on subcellular localization that may be relevant to cancer biology and therapeutic responses.

What approaches can resolve contradictory results in PRDX3 expression analysis across different cancer types?

Resolving contradictory results in PRDX3 expression analysis requires systematic methodological approaches:

• Standardization of detection methods:

  • Use consistent antibody clones and dilutions across studies (PRDX3 antibody at 1:5000-1:50000 for WB, 1:50-1:500 for IHC)

  • Implement quantitative Western blotting with internal loading controls

  • Establish standardized scoring systems for IHC (0=negative, 1=weak, 2=moderate, 3=strong)

  • Include technical replicates and biological replicates

• Multi-level analysis approach:

  • Examine PRDX3 at protein level (Western blot, IHC) and mRNA level (RT-qPCR, RNA-seq)

  • Compare results to identify post-transcriptional regulation

  • Include proteomic approaches for unbiased detection

• Context-specific stratification:

  • Stratify samples by cancer subtypes, stages, and grades

  • Account for treatment history when analyzing patient samples

  • Consider microenvironmental factors that may influence PRDX3 expression

• Technical validation:

  • Use multiple antibodies targeting different epitopes

  • Include appropriate positive and negative controls

  • Validate antibody specificity using PRDX3 knockout or knockdown samples

How reliable is PRDX3 as a prognostic biomarker in cancer research?

The reliability of PRDX3 as a prognostic biomarker varies by cancer type and requires rigorous assessment methodology:

• Statistical validation approaches for biomarker assessment:

  • Perform univariate survival analysis (Kaplan-Meier method with log-rank test)

  • Conduct multivariate analysis (Cox proportional hazards model) to adjust for confounding factors

  • Calculate hazard ratios with 95% confidence intervals

  • Determine optimal cut-off values using ROC curve analysis

• Standardized tissue microarray (TMA) analysis:

  • Construct TMAs from well-characterized patient cohorts with complete clinical follow-up

  • Stain with validated PRDX3 antibody (1:50-1:500)

  • Implement digital pathology for quantitative scoring

  • Use H-score method (intensity × percentage of positive cells) for standardization

• Meta-analysis methodology:

  • Combine data from multiple independent cohorts

  • Assess heterogeneity using statistical methods

  • Calculate pooled hazard ratios

  • Evaluate publication bias

These findings suggest PRDX3 may be a valuable prognostic biomarker, particularly in advanced ovarian cancer, but its utility may vary based on histological subtype, grade, and stage .

What protocols are optimal for using PRDX3 antibody to investigate oxidative stress-induced apoptosis in cancer cells?

Investigating oxidative stress-induced apoptosis with PRDX3 antibody requires these optimized protocols:

• Oxidative stress induction and monitoring:

  • Treat cells with oxidizing agents (H₂O₂, tert-butyl hydroperoxide) at various concentrations

  • Measure intracellular ROS using fluorescent probes (DCFDA, MitoSOX Red)

  • Monitor mitochondrial membrane potential using JC-1 dye

  • Track PRDX3 expression by Western blot (1:5000-1:50000 dilution)

• PRDX3 expression manipulation studies:

  • Generate stable or transient PRDX3 knockdown using siRNA/shRNA

  • Create PRDX3 overexpression models via transfection

  • Verify expression changes by Western blot

  • Compare oxidative stress responses between modified and control cells

• Apoptosis assessment methodology:

  • Measure early apoptosis by Annexin V/PI staining and flow cytometry

  • Detect caspase activation using fluorogenic substrates or Western blot

  • Assess PARP cleavage by Western blot

  • Perform TUNEL assay for DNA fragmentation

• Signaling pathway investigation:

  • Examine pro-apoptotic (Bax, Bak) and anti-apoptotic (Bcl-2, Bcl-xL) protein levels

  • Assess cytochrome c release from mitochondria by subcellular fractionation

  • Investigate activation of stress response pathways

Research has demonstrated that PRDX3 plays a crucial role in regulating oxidation-induced apoptosis in cancer cells. In antiandrogen-resistant prostate cancer cells, PRDX3 is upregulated at the protein level, and these cells show resistance to H₂O₂-induced apoptosis through a failure to activate pro-apoptotic pathways . Notably, knockdown of PRDX3 restored H₂O₂ sensitivity, suggesting that PRDX3 has an essential role in regulating oxidation-induced apoptosis in these cells . These findings indicate PRDX3 may have potential as a therapeutic target in castrate-independent prostate cancer.

How can PRDX3 antibody be used to evaluate novel redox-modulating cancer therapies?

PRDX3 antibody can be instrumental in evaluating novel redox-modulating cancer therapies through these methodological approaches:

• Baseline and post-treatment expression analysis:

  • Collect tumor samples before and after treatment

  • Perform IHC with PRDX3 antibody (1:50-1:500)

  • Quantify expression changes using digital image analysis

  • Correlate with clinical response metrics

• Redox status assessment:

  • Detect PRDX3 oxidation state using non-reducing Western blots

  • Distinguish between reduced monomeric and oxidized dimeric forms

  • Monitor hyperoxidation using antibodies specific for hyperoxidized peroxiredoxins

  • Track regeneration of active PRDX3 by the thioredoxin system

• Combination therapy evaluation:

  • Test redox-modulating drugs in combination with standard chemotherapies

  • Assess PRDX3 expression and oxidation state after treatment

  • Analyze synergistic effects on cancer cell death

  • Determine optimal sequencing and dosing

• Patient stratification marker development:

  • Correlate PRDX3 expression patterns with treatment outcomes

  • Develop predictive models for patient stratification

  • Define cutoff values for high versus low PRDX3 expression

  • Validate in independent patient cohorts

Studies have shown that PRDX3 is associated with drug resistance in various cancers. In ovarian cancer, PRDX3 expression was significantly higher in platinum-resistant serous ovarian cancer compared to platinum-sensitive counterparts . In prostate cancer, PRDX3 is upregulated in antiandrogen-resistant cell lines and contributes to resistance against oxidative stress-induced apoptosis . These findings suggest that monitoring PRDX3 expression and modulating its activity could be valuable strategies in developing and evaluating new cancer therapies targeting redox pathways.

How can different peroxiredoxin family members be distinguished when using antibodies in experimental settings?

Distinguishing between peroxiredoxin family members requires careful experimental design:

• Antibody selection strategies:

  • Choose antibodies raised against unique regions with lowest sequence homology

  • Verify specificity against recombinant proteins of all PRDX family members

  • Use monoclonal antibodies targeting isoform-specific epitopes

  • Validate by Western blot of tissues with differential PRDX expression

• Western blot optimization for improved discrimination:

  • Use higher percentage gels (12-15%) to maximize separation

  • Apply extended run times to resolve closely migrating bands

  • Compare molecular weights: PRDX3 (26-28 kDa) vs. other PRDXs

  • Consider 2D-electrophoresis for enhanced separation

• Subcellular localization profiling:

  • Use confocal microscopy with co-localization markers

  • PRDX3: primarily mitochondrial (co-localize with MitoTracker)

  • PRDX1/2: predominantly cytosolic

  • PRDX4: endoplasmic reticulum

  • PRDX5: multiple compartments (mitochondria, peroxisomes, cytosol)

  • PRDX6: cytosolic and lysosomal

• Genetic knockdown validation controls:

  • Generate knockdown cells for each PRDX isoform

  • Confirm antibody specificity by diminished signal in appropriate knockdown

  • Use as negative controls in immunoblotting and immunostaining

The peroxiredoxin family consists of six members (PRDX1-6) with distinct subcellular localizations but similar molecular weights and functions . PRDX3 is distinguished by its predominant mitochondrial localization , which can be used as a key differentiating feature. When performing immunostaining, co-localization with mitochondrial markers can confirm PRDX3 specificity. For Western blotting, although the molecular weights are similar, careful optimization of gel conditions can allow separation, with PRDX3 typically appearing at 26-28 kDa .

What factors affect PRDX3 detection in oxidative stress experiments?

Multiple factors can affect PRDX3 detection in oxidative stress experiments:

• Oxidation-induced structural changes:

  • PRDX3 forms dimers and oligomers under oxidizing conditions

  • Use non-reducing SDS-PAGE to preserve these structures

  • Include both reducing and non-reducing conditions for comprehensive analysis

  • Consider band shift: monomeric (26-28 kDa) vs. dimeric (~55 kDa) forms

• Sample preparation considerations:

  • Add alkylating agents (NEM, IAA) immediately during lysis to prevent artificial oxidation

  • Include antioxidants in lysis buffers

  • Maintain cold temperature throughout processing

  • Minimize handling time between cell harvesting and protein extraction

• Hyperoxidation effects:

  • Strong oxidants can cause sulfinic/sulfonic acid formation on catalytic cysteine

  • These modifications are not reversed by reducing agents

  • Use antibodies specific for hyperoxidized peroxiredoxins to detect this form

  • Consider alterations in antibody epitope recognition due to hyperoxidation

• Experimental design controls:

  • Include positive controls (H₂O₂-treated cells) and negative controls

  • Use time-course experiments to capture dynamic redox changes

  • Implement parallel orthogonal methods (e.g., mass spectrometry)

  • Consider measuring PRDX3 activity alongside protein levels

Research has shown that PRDX3's function and detection can be significantly affected by its redox state . Under oxidative stress conditions, PRDX3 can undergo various oxidative modifications, including reversible disulfide bond formation and irreversible hyperoxidation. These modifications can affect antibody binding and protein migration patterns in gels, potentially leading to misinterpretation of results if not properly controlled.

How might PRDX3 antibodies be applied in developing therapeutic strategies targeting cancer redox mechanisms?

PRDX3 antibodies hold potential for developing novel cancer therapeutic strategies:

• Target validation approaches:

  • Use PRDX3 antibodies to evaluate expression across cancer types and patient samples

  • Correlate expression with treatment response and patient outcomes

  • Identify high-expression subtypes that may benefit from PRDX3-targeted approaches

  • Validate subcellular localization in patient-derived samples

• Drug development applications:

  • Screen for compounds that modulate PRDX3 expression or activity

  • Develop cell-based high-throughput assays using PRDX3 antibodies

  • Monitor drug effects on PRDX3 oxidation state and protein levels

  • Identify synthetic lethal interactions with PRDX3 inhibition

• Combination therapy optimization:

  • Evaluate PRDX3 modulation in response to standard chemotherapies

  • Identify synergistic combinations targeting redox homeostasis

  • Monitor treatment-induced changes in PRDX3 localization and modification

  • Develop rational sequencing strategies based on redox dynamics

• Precision medicine applications:

  • Stratify patients based on PRDX3 expression levels

  • Develop companion diagnostics using PRDX3 antibodies

  • Monitor treatment response via changes in PRDX3 expression/oxidation

  • Guide therapy selection based on PRDX3 status