PRDX3 Monoclonal Antibody

What is PRDX3 and what are its primary cellular functions?

PRDX3 (Peroxiredoxin 3) is a mitochondrial hydrogen peroxide scavenger that belongs to the ahpC/TSA family. It plays a crucial role in redox regulation within cells by protecting radical-sensitive enzymes from oxidative damage. PRDX3 is predominantly localized in mitochondria and functions to reduce hydrogen peroxide continuously generated in this organelle. The protein has a calculated molecular weight of 27 kDa, though it is typically observed at 26-28 kDa in experimental conditions .

Recent research has established that PRDX3 is frequently upregulated during tumorigenesis and cancer progression, particularly in breast cancer, where it regulates cellular signaling pathways associated with Matrix Metalloproteinase-1 (MMP-1) expression and activity . The protein's role in maintaining mitochondrial redox homeostasis makes it a critical factor in cellular stress responses and potential therapeutic target in various pathological conditions.

What types of PRDX3 antibodies are available for research applications?

Two primary types of PRDX3 antibodies are available for research:

• Polyclonal antibodies (e.g., 10664-1-AP): These antibodies recognize multiple epitopes on the PRDX3 protein, offering high sensitivity. They are produced in rabbits and purified through antigen affinity methods .

• Monoclonal antibodies (e.g., 66810-1-Ig): These offer high specificity by recognizing a single epitope. PRDX3 monoclonal antibodies are typically mouse-derived (IgG2a isotype) and purified through Protein A purification methods .

Both antibody types can be used across multiple applications including Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF)/Immunocytochemistry (ICC), with validated reactivity against human, mouse, and rat samples, depending on the specific antibody .

What is the recommended storage and handling protocol for PRDX3 antibodies?

For optimal performance and shelf-life of PRDX3 antibodies:

• Store antibodies at -20°C in their provided buffer (typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3) .

• Antibodies are stable for one year after shipment when stored properly.

• Aliquoting is unnecessary for -20°C storage, reducing the risk of contamination and freeze-thaw cycles.

• Some preparations (20μl sizes) contain 0.1% BSA as a stabilizer .

• When working with the antibody, minimize freeze-thaw cycles and keep on ice during experimental procedures.

• Return to -20°C promptly after use to maintain antibody integrity and performance.

What are the recommended dilutions for different applications of PRDX3 antibodies?

The optimal dilution varies by antibody type and application. Based on validated data, the following dilutions are recommended:

For PRDX3 Polyclonal Antibody (10664-1-AP):

For PRDX3 Monoclonal Antibody (66810-1-Ig):

These dilutions should be optimized for each experimental system to obtain the best results . Sample-dependent variations may require adjustment of these recommended ranges.

How should I optimize Western blot protocols for PRDX3 detection?

For optimal Western blot detection of PRDX3:

• Sample preparation: Use appropriate lysis buffers that preserve protein integrity. For mitochondrial proteins like PRDX3, consider mitochondrial isolation protocols if studying mitochondria-specific expression or modifications.

• Loading controls: Include mitochondrial markers when analyzing PRDX3 in mitochondrial fractions.

• Expected molecular weight: Look for bands at 26-28 kDa, which is the observed molecular weight of PRDX3 . Be aware that a smaller form (~16 kDa, termed PRDX3-S) may appear under oxidative stress conditions or in certain disease states .

• Positive controls: Use lysates from validated cell lines including HeLa, HEK-293, MCF-7, or Jurkat cells, which have been confirmed to express detectable levels of PRDX3 .

• Antibody dilution: Start with the recommended dilution range and adjust as needed. The monoclonal antibody (66810-1-Ig) requires higher dilution (1:20000-1:100000) compared to the polyclonal antibody (1:5000-1:50000) .

• Signal development: For this well-expressed protein, shorter exposure times may be sufficient to detect the specific signal while minimizing background.

What are the best practices for immunohistochemistry with PRDX3 antibodies?

For effective immunohistochemical detection of PRDX3:

• Antigen retrieval: Use TE buffer at pH 9.0 for optimal results. Alternatively, citrate buffer at pH 6.0 may be used, though potentially with lower signal intensity .

• Tissue specificity: PRDX3 antibodies have been validated on human liver and lung cancer tissues, making these appropriate positive controls .

• Dilution optimization: For polyclonal antibodies, start with 1:50-1:500 dilution; for monoclonal antibodies, use 1:1000-1:4000 dilution .

• Signal interpretation: Expect predominantly mitochondrial localization in normal tissues, with potential altered distribution patterns in cancer tissues.

• Controls: Include both positive tissue controls (liver or lung cancer) and negative controls (omitting primary antibody) to confirm specificity.

• Counterstaining: Use mitochondrial markers in parallel sections to confirm the mitochondrial localization of PRDX3.

How does PRDX3 contribute to cancer progression mechanisms?

PRDX3's role in cancer progression is multifaceted:

• Regulation of cell migration and invasion: PRDX3 significantly impacts the metastatic potential of cancer cells. Research using shRNA-mediated gene silencing of PRDX3 demonstrated inhibition of cell migration and invasion in triple-negative breast cancer cell lines. Conversely, PRDX3 overexpression enhanced migration and invasion capabilities .

• MMP-1 regulation: PRDX3 upregulates Matrix Metalloproteinase-1 (MMP-1), a crucial enzyme in extracellular matrix degradation during cancer metastasis. Both mRNA expression and active MMP-1 secretion levels are positively correlated with PRDX3 expression levels. Specifically, PRDX3 knockdown reduced active MMP-1 activity by 2.4-fold, while PRDX3 overexpression increased it by 4.8-fold .

• Signaling pathway modulation: PRDX3 activates the ERK signaling pathway, leading to increased AP-1 transcriptional activity (>3.3-fold induction) and c-Jun phosphorylation. This activation directly contributes to MMP-1 transcription. Importantly, ERK inhibition using SCH772984 reduced MMP-1 expression in PRDX3-overexpressing cells, confirming this regulatory mechanism .

• Clinical correlation: Immunohistochemical analysis of breast cancer tissues revealed a positive correlation between PRDX3 and MMP-1 expression in both epithelial and stromal components, supporting the laboratory findings in human disease .

These findings suggest that PRDX3 could be a potential therapeutic target in triple-negative breast cancer, particularly through inhibition of the ERK signaling pathway to prevent tumor metastasis.

What is the relationship between PRDX3 and oxidative stress in mitochondria?

PRDX3's primary function is to maintain redox homeostasis within mitochondria:

These findings highlight PRDX3's critical role in protecting mitochondria from oxidative damage and maintaining cellular redox homeostasis, with implications for diseases characterized by oxidative stress.

How does PRDX3 interact with other proteins in cellular signaling networks?

PRDX3 engages in several important protein-protein interactions:

• FANCG interaction: PRDX3 physically interacts with FANCG (a Fanconi anemia gene product), as demonstrated through both yeast two-hybrid analysis and coimmunoprecipitation assays. This interaction is diminished in FANCG protein containing the patient-associated G546R mutation .

• ERK signaling pathway components: PRDX3 promotes the phosphorylation of MAPK and other serine/threonine kinases involved in cancer metastasis. This activation appears to be part of PRDX3's role in promoting cell invasion and migration .

• AP-1 transcription complex: PRDX3 overexpression leads to increased phosphorylation of c-Jun, a major component of the AP-1 transcription complex, which regulates MMP-1 expression. This provides a mechanistic link between PRDX3 and downstream gene expression changes .

• Transcription factor regulation: PRDX3 affects multiple transcriptional activities, including Nrf2/Nrf1, NF-kB, HIF1, CBF/NF-Y-YY1, HSF1, AP1, and AhR, suggesting broad influence on cellular gene expression programs .

Understanding these interaction networks is crucial for comprehending how PRDX3 functions in different cellular contexts and how it might be targeted therapeutically in conditions like cancer or oxidative stress-related disorders.

Why might I observe multiple bands in Western blots using PRDX3 antibodies?

Multiple bands in PRDX3 Western blots can occur for several research-relevant reasons:

• Oxidative stress-induced cleavage: A smaller form of PRDX3 (PRDX3-S, approximately 16 kDa) can appear under oxidative stress conditions. This was observed in FA-G mutant cells and in FANCG-corrected cells after exposure to 1 mM H₂O₂ . This form may represent a stress-induced cleavage product with potentially altered function.

• Post-translational modifications: PRDX3 undergoes various modifications including oxidation states that can alter its mobility on SDS-PAGE. The peroxiredoxin catalytic cycle involves formation of disulfide bonds that may not be fully reduced during sample preparation.

• Mitochondrial processing: As a mitochondrial protein, PRDX3 contains an N-terminal mitochondrial targeting sequence that is cleaved upon import into mitochondria. Incomplete processing can result in multiple bands representing precursor and mature forms.

• Antibody cross-reactivity: Some antibodies may recognize other peroxiredoxin family members (PRDX1-6) due to sequence homology. Checking the specific epitope recognized by your antibody can help identify potential cross-reactivity.

To distinguish between these possibilities, consider:

• Including both reducing and non-reducing conditions

• Comparing mitochondrial and whole-cell lysates

• Using positive controls (e.g., lysates from HeLa, HEK-293, or MCF-7 cells)

• Testing cells under normal and oxidative stress conditions

How can I validate the specificity of my PRDX3 antibody results?

For rigorous validation of PRDX3 antibody specificity:

These approaches collectively provide strong evidence for antibody specificity and result reliability.

How is PRDX3 involved in the mechanism of cancer metastasis?

PRDX3's role in cancer metastasis involves several coordinated molecular mechanisms:

• ERK-MMP1 axis activation: PRDX3 promotes ERK signaling pathway activation, which in turn activates the AP-1 transcription complex. This leads to increased transcription of MMP-1, a matrix metalloproteinase that degrades extracellular matrix components and facilitates cancer cell invasion .

• Enhancement of cell migration and invasion: Experimental evidence demonstrates that PRDX3 overexpression significantly enhances migration and invasion in breast cancer cells, while its knockdown inhibits these processes. These are fundamental capabilities required for metastatic progression .

• Transcriptional regulation network: PRDX3 affects multiple transcription factors including AP-1, Nrf2/Nrf1, NF-kB, and HIF1, suggesting it may coordinate a broader pro-metastatic gene expression program beyond MMP-1 .

• Clinical correlation: The positive correlation between PRDX3 and MMP-1 expression in both epithelial and stromal components of breast cancer tissues suggests this mechanism is relevant in human disease .

• Therapeutic targeting potential: Inhibition of ERK signaling reduced MMP-1 expression in PRDX3-overexpressing cells, indicating that targeting this pathway may represent a strategy to inhibit PRDX3-mediated metastasis in triple-negative breast cancer .

This mechanistic understanding provides several potential intervention points for anti-metastatic therapies, particularly in aggressive cancer types where conventional treatments may be less effective.

What are the implications of PRDX3 mislocalization in disease states?

PRDX3 mislocalization has significant implications for cellular function and disease pathology:

These findings highlight how protein mislocalization, beyond simple expression changes, can contribute to disease mechanisms and potentially provide diagnostic or therapeutic insights for conditions involving mitochondrial dysfunction or oxidative stress.