PRDX4 Antibody

What molecular weight should I expect when detecting PRDX4 in Western blot?

PRDX4 typically appears at 30-32 kDa in Western blot applications, though the observed molecular weight may vary slightly between different cell lines and tissue samples. The calculated molecular weight is approximately 31 kDa, while the observed molecular weight is typically 27-30 kDa . Some researchers also report detecting a band at 27 kDa, which potentially represents a cleaved form of PRDX4 due to the presence of a 39 amino acid signal peptide . When using reducing conditions, PRDX4 is generally detected as a monomer, while under non-reducing conditions, it may appear as dimers or higher-order complexes, reflecting its functional state as a pentamer of dimers .

What are the recommended dilutions for different applications of PRDX4 antibodies?

The optimal dilution varies by application and specific antibody. Based on validated protocols, the following ranges are recommended:

It is recommended to optimize these dilutions for each specific experimental system and sample type . For Western blot detection of PRDX4, some researchers have found optimal results using 1 μg/mL of antibody when probing PVDF membranes .

What cell lines have been validated for PRDX4 antibody detection?

Multiple cell lines have been validated for detecting endogenous PRDX4 expression:

Human cancer cell lines such as HEK293, K562, and HeLa consistently show strong PRDX4 expression detectable by various antibodies . When establishing new detection protocols, these cell lines can serve as positive controls.

How can I distinguish between the membrane-bound and secreted forms of PRDX4?

PRDX4 exists in two forms: a membrane-binding 31-kDa protein and a processed 27-kDa secretory form . To distinguish between these forms:

• Subcellular fractionation: Separate cell lysate into cytosolic, membrane, and secreted fractions before Western blot analysis.

• Antibody selection: Use antibodies targeting different epitopes - those recognizing the N-terminal region can detect differences in signal peptide processing.

• Molecular weight comparison: The 27 kDa band observed in some Western blots represents the cleaved/secretory form, while the 30-32 kDa band corresponds to the full-length protein with the signal peptide .

• Secretion analysis: Compare cell lysate with concentrated culture medium to identify secreted PRDX4.

Research indicates that PRDX4 is initially synthesized as a membrane-binding 31-kDa protein and processed into a 27-kDa secretory form during cellular processes such as spermatogenesis .

What are optimal antigen retrieval methods for PRDX4 IHC in FFPE tissues?

For optimal PRDX4 detection in formalin-fixed paraffin-embedded (FFPE) tissues, antigen retrieval is critical:

• TE buffer method: The primary recommended method is heat-induced epitope retrieval with TE buffer at pH 9.0, which has been validated for human cancer tissues including colon, pancreas, and liver cancers .

• Citrate buffer alternative: As an alternative, citrate buffer at pH 6.0 can be used, though this may yield different staining intensity .

• Incubation time and temperature: For human liver cancer tissue, researchers have successfully used PRDX4 antibodies at 0.3 μg/mL for 1 hour at room temperature followed by appropriate secondary antibody detection systems .

• Visualization systems: DAB (diaminobenzidine) chromogen with hematoxylin counterstaining provides good contrast for evaluating PRDX4 expression patterns in cancer cells .

In gastric cancer research, PRDX4 expression scoring has been standardized based on the percentage of positive cells: Score 0 (<1%), score 1 (1–25%), score 2 (25–49%), and score 3 (50–74%) .

How can I validate PRDX4 antibody specificity in my experimental system?

Validating antibody specificity is crucial for accurate PRDX4 research:

• PRDX4 knockdown/knockout controls: Use shRNA-mediated knockdown or CRISPR/Cas9 knockout systems. Research has validated PRDX4 antibody specificity using shRNA in cell lines like AGS and MKN28 . Alternatively, PRDX4 knockout mouse models can serve as negative controls for antibody specificity .

• Isoform specificity testing: Confirm that your antibody is specific for PRDX4 and does not cross-react with other peroxiredoxin family members. Some antibodies are specifically tested for non-cross-reactivity with other PRDX isoforms .

• Peptide competition assay: Pre-incubate the antibody with a synthetic blocking peptide corresponding to the immunogen sequence . A significant reduction in signal indicates specificity.

• Multiple antibody validation: Compare results using antibodies raised against different epitopes of PRDX4. Consistent detection patterns across different antibodies increase confidence in specificity.

• Western blot molecular weight verification: Confirm that the detected band matches the expected molecular weight of PRDX4 (approximately 30 kDa) .

How is PRDX4 expression associated with cancer progression and prognosis?

PRDX4 has emerged as a potential biomarker in cancer research, particularly in gastric cancer:

• Prognostic value: Immunohistochemical analysis has demonstrated that PRDX4 overexpression is significantly associated with poor prognosis in gastric cancer patients. The PRDX4-overexpressing group showed significantly worse survival than the PRDX4-underexpression group (P<0.01) .

• Correlation with clinical parameters: PRDX4 overexpression correlates with:

  • Undifferentiated cancer types (poorly differentiated and signet ring cell type)

  • Advanced gastric cancer (T2-T4)

  • Metastatic lymph nodes (N≥1)

  • Higher TNM stage (stage II~IV)

  • Higher rates of cancer-related death (76.1% vs. 23.9%) and recurrence (71.6% vs. 28.4%)

• Functional mechanisms: In gastric cancer models, knockdown of PRDX4 expression by shRNA caused a significant decrease in cancer cell invasion, while overexpression of PRDX4 in PRDX4-depleted cancer cells promoted migration and invasion .

• Molecular pathways: PRDX4 influences the epithelial-mesenchymal transition (EMT) pathway - knockdown of PRDX4 increased E-cadherin expression and decreased snail and slug expression, key regulators of EMT .

• Protein interactions: PRDX4 has been identified as an important interacting protein of TXNDC5, a gastric cancer-promoting gene. This interaction may promote tumor progression by regulating the tumor immune microenvironment .

What are the key considerations when designing co-immunoprecipitation experiments to study PRDX4 protein interactions?

When investigating PRDX4 protein interactions through co-immunoprecipitation:

• Antibody selection: Choose antibodies validated specifically for immunoprecipitation applications. For PRDX4, successful IP has been demonstrated using 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate .

• Cell line selection: HepG2 cells have been validated for PRDX4 immunoprecipitation and can serve as a reliable model system .

• Buffer optimization: Consider the redox-sensitive nature of PRDX4 when designing lysis and IP buffers. Include appropriate reducing agents or oxidation inhibitors to preserve physiological interactions.

• Control experiments:

  • Use IgG from the same species as the PRDX4 antibody as a negative control

  • Perform reverse co-IP when studying specific interactions (e.g., PRDX4-TXNDC5)

  • Include input controls (5-10% of lysate used for IP)

• Crosslinking considerations: For transient or weak interactions, consider using crosslinking reagents prior to cell lysis to stabilize protein complexes.

• Validation methods: Confirm interactions using orthogonal methods such as proximity ligation assay or fluorescence resonance energy transfer (FRET).

How can I differentiate between various redox states of PRDX4 in experimental samples?

PRDX4's function depends on its redox state, which can be analyzed using specific techniques:

• Non-reducing vs. reducing SDS-PAGE: Under non-reducing conditions, different redox states of PRDX4 can be separated based on intramolecular and intermolecular disulfide bonds. Compare samples run on gels with and without reducing agents like DTT or β-mercaptoethanol.

• Alkylation of free thiols: Prior to cell lysis, treat samples with thiol-blocking agents (e.g., N-ethylmaleimide) to prevent artificial oxidation during sample preparation.

• Antibodies to specific redox forms: Some specialized antibodies can distinguish between reduced, oxidized, or hyperoxidized forms of peroxiredoxins based on the state of the catalytic cysteine residue (Cys124 in PRDX4).

• 2D redox proteomics: Combine isoelectric focusing with SDS-PAGE to separate PRDX4 based on both charge and mass differences resulting from oxidative modifications.

• Mass spectrometry analysis: Use targeted mass spectrometry approaches to identify specific oxidative modifications on PRDX4, including disulfide formation, sulfinic acid (SO₂H), or sulfonic acid (SO₃H) modifications of cysteine residues.

What is the role of PRDX4 in testicular function and fertility research?

PRDX4 has specific functions in male reproductive biology:

• Testis-specific variant: A testis-specific PRDX4 variant transcript (PRDX4t) exists that lacks the conventional exon 1 encoding the signal peptide. Instead, it carries an alternative exon 1 transcribed from an upstream promoter in a testis-specific manner, resulting in cytosolic localization rather than ER localization .

• Knockout studies: Interestingly, knockout studies found that mice lacking PRDX4t underwent normal spermatogenesis and showed no overt abnormalities in the testis. Male PRDX4t knockout mice maintained normal fertility when mated with wild-type females .

• Compensatory mechanisms: Double knockout mice lacking both PRDX4 and PRDX4t remained fertile, with protein levels of glutathione peroxidase 4 (GPX4) significantly increased in the testis and caput epididymis compared to wild-type mice, suggesting compensatory mechanisms .

• Acrosome formation: PRDX4 is associated with acrosome formation during rat spermatogenesis and has a protective role in the male reproductive tract. The protein is initially synthesized as a membrane-binding form and processed into a secretory form during spermatogenesis .

How can I optimize PRDX4 antibody use for multiplexed immunofluorescence studies?

For multiplexed detection of PRDX4 alongside other proteins:

• Antibody compatibility: Choose PRDX4 antibodies raised in different host species than antibodies against other target proteins to avoid cross-reactivity in multiplexed staining.

• Validated protocols: PRDX4 has been successfully visualized in human cell lines using immunofluorescence. For example, in HeLa cells, researchers have used Mouse Anti-Human PRDX4 Monoclonal Antibody at 10 μg/mL for 3 hours at room temperature, followed by fluorophore-conjugated secondary antibodies .

• Subcellular localization: PRDX4 shows specific staining localized to the cytoplasm in validated immunofluorescence applications . Consider this when designing co-localization studies with other proteins.

• Sequential staining approach: For challenging multiplex panels, consider sequential staining with appropriate blocking steps between antibody applications.

• Controls for autofluorescence: Include unstained controls and single-stained controls to account for tissue autofluorescence and spectral overlap when using multiple fluorophores.

• Signal amplification: For low-abundance targets, consider using signal amplification systems like tyramide signal amplification while keeping PRDX4 detection conventional to maintain optimal signal-to-noise ratios.

What are the considerations for using PRDX4 antibodies in cancer tissue microarray (TMA) analysis?

When analyzing PRDX4 expression in cancer tissue microarrays:

• Scoring system standardization: Establish a consistent scoring system for PRDX4 expression. Previous research has used a percentage-based system: Score 0 (<1%), score 1 (1–25%), score 2 (25–49%), and score 3 (50–74%) .

• Prognostic grouping: Consider categorizing samples into PRDX4-overexpression (scores 2+ and 3+) and PRDX4-underexpression (scores 0 and 1+) groups for survival analysis and clinical correlation .

• Clinical parameter correlation: Analyze PRDX4 expression in relation to:

  • Tumor differentiation

  • Tumor invasion depth

  • Lymph node metastasis

  • TNM staging

  • Treatment response

  • Survival outcomes

• Multivariate analysis: Include PRDX4 expression alongside established prognostic factors in Cox proportional hazard models to determine independent prognostic value .

• Antibody validation on whole sections: Before proceeding with TMA analysis, validate antibody performance on whole tissue sections to confirm staining patterns and optimize protocols.

• Combination with other markers: Consider analyzing PRDX4 alongside interacting proteins like TXNDC5 or markers of oxidative stress to develop more comprehensive prognostic panels .

How can PRDX4 antibodies be utilized in studying oxidative stress pathways in disease models?

PRDX4 plays important roles in cellular redox regulation and stress responses:

• Oxidative stress monitoring: PRDX4 antibodies can be used to monitor changes in expression levels in response to oxidative stress inducers, providing insights into cellular antioxidant responses.

• Cancer research applications: In cancer research, PRDX4 has been shown to influence tumor progression by modulating the antioxidant capacity within cells . Antibodies can help track these changes across different cancer types and stages.

• NF-κB pathway investigation: PRDX4 regulates the activation of NF-κB in the cytosol by modulating I-κB-α phosphorylation . Combining PRDX4 antibodies with phospho-specific I-κB-α antibodies can help elucidate this regulatory mechanism.

• ER stress studies: As PRDX4 is involved in endoplasmic reticulum function, antibodies can be used in combination with other ER stress markers to understand the relationship between oxidative stress and ER stress pathways.

• Therapeutic response monitoring: Changes in PRDX4 levels or localization following treatment with antioxidants or redox-modulating drugs can provide insights into therapeutic mechanisms and efficacy.

What are the current challenges and solutions in detecting post-translational modifications of PRDX4?

Detecting post-translational modifications (PTMs) of PRDX4 presents several challenges:

• Oxidation state-specific detection: The redox-active cysteine (Cys124) in PRDX4 can exist in different oxidation states. Specialized approaches include:

  • Differential alkylation strategies to trap specific redox states

  • Development of antibodies specific to oxidized forms

  • Mass spectrometry-based redox proteomics

• Phosphorylation analysis: PRDX4 function may be regulated by phosphorylation. Approaches include:

  • Phospho-specific antibodies (when available)

  • Phospho-enrichment prior to Western blot analysis

  • Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated forms

• Other PTMs: For ubiquitination, SUMOylation, or acetylation:

  • Immunoprecipitate PRDX4 followed by Western blotting with PTM-specific antibodies

  • Use proteomic approaches with enrichment for specific modifications

  • Employ cell models with inhibitors of specific PTM-regulating enzymes

• Low abundance challenges: Many PTMs occur on only a fraction of the total protein pool. Solutions include:

  • Subcellular fractionation to enrich modified forms

  • Proximity ligation assays for in situ detection of specific modified forms

  • Super-resolution microscopy to detect co-localization with PTM-regulating enzymes

How does PRDX4 expression in cancer models compare with other peroxiredoxin family members?

Understanding the differential expression of peroxiredoxin family members in cancer:

• Subcellular localization differences: While PRDX4 is primarily associated with the ER and secretory pathway, other family members have distinct localizations: PRDX1, PRDX2, and PRDX6 in cytosol, PRDX3 in mitochondria, and PRDX5 in multiple compartments including peroxisomes, mitochondria, and cytosol . This necessitates careful subcellular fractionation when comparing family members.

• Cancer-specific expression patterns: PRDX4 overexpression has been specifically linked to poor prognosis in gastric cancer . When designing comparative studies:

  • Use antibodies verified for non-cross-reactivity with other PRDX family members

  • Consider multiplex immunofluorescence to visualize multiple family members simultaneously

  • Correlate expression patterns with subcellular oxidative stress markers

• Functional redundancy vs. specificity: Despite structural similarities, PRDX family members show specific functions. When investigating PRDX4:

  • Consider knockdown/knockout of multiple family members to assess compensatory mechanisms

  • Analyze co-expression patterns in clinical samples

  • Investigate interaction partners unique to PRDX4 versus other family members

• Secreted vs. intracellular forms: Unlike most other PRDXs, PRDX4 has a significant secreted component, allowing examination in liquid biopsies and extracellular studies .