PRDX3 Mouse mAb: Selection, Application & Protocol Guide

PRDX3 Mouse mAb is a mouse-derived monoclonal antibody specifically targeting human Peroxiredoxin-3 (PRDX3), a mitochondria-localized thioredoxin-dependent peroxidase critical for regulating intracellular reactive oxygen species and maintaining mitochondrial redox homeostasis.

Product SKU

Ab349216

Antibody Format

Mouse Monoclonal Antibody (mAb)

Target Protein

PRDX3 (Peroxiredoxin-3), a mitochondria-resident 2-Cys peroxiredoxin involved in H₂O₂ scavenging

Primary Applications

Western Blot (WB), Immunofluorescence (IF), Immunohistochemistry (IHC) — confirm suitability in datasheet

Recommended Storage

Store at –20 °C; avoid repeated freeze-thaw cycles; refer to product datasheet for exact formulation details

Host / Isotype

Mouse; isotype to be confirmed on product datasheet

Subcellular Localization of Target

Mitochondrial matrix

Background: PRDX3 and Mitochondrial Redox Biology

Peroxiredoxin-3 (PRDX3) is a member of the 2-Cys peroxiredoxin subfamily and is the predominant peroxiredoxin isoform localized to the mitochondrial matrix. It functions as a thioredoxin-2-dependent peroxidase, reducing hydrogen peroxide (H₂O₂) and organic hydroperoxides generated as by-products of the mitochondrial electron transport chain.

PRDX3 exists as a homodimer under normal redox conditions and forms higher-order oligomers (decamers) under oxidative stress. The catalytic cycle involves oxidation of the peroxidatic cysteine residue to sulfenic acid, which then forms an intramolecular disulfide with the resolving cysteine; this disulfide is subsequently reduced by thioredoxin-2 (TXN2), regenerating the active enzyme.

Because PRDX3 governs mitochondrial H₂O₂ levels, it plays a key role in redox signaling, apoptosis regulation, and metabolic control. Dysregulation of PRDX3 expression has been reported in cancer, neurodegeneration, and metabolic disorders, making it a relevant biomarker and therapeutic target in current research.

Antibody Specifications and Validated Applications

Product: PRDX3 Mouse mAb (SKU: Ab349216, Absea catalog reference KC-8488). This is a mouse monoclonal antibody raised against human PRDX3. Monoclonal format ensures lot-to-lot consistency and high epitope specificity compared with polyclonal alternatives.

Recommended applications typically for anti-PRDX3 monoclonal antibodies include:

• Western Blot (WB): Detection of PRDX3 at the expected molecular weight of approximately 28–30 kDa under reducing conditions (note: dimeric forms may appear at ~56–60 kDa under non-reducing conditions).
• Immunofluorescence (IF): Visualization of mitochondrial localization patterns in cultured cells using confocal or epifluorescence microscopy.
• Immunohistochemistry (IHC): Detection of PRDX3 expression in formalin-fixed paraffin-embedded (FFPE) tissue sections.

Always consult the current product datasheet for validated dilutions, positive control cell lines, and cross-reactivity data, as these specifications are lot-dependent.

Recommended Protocols

Western Blot Protocol

1. Sample preparation: Lyse cells or tissue in RIPA buffer supplemented with protease inhibitor cocktail. Quantify protein by BCA or Bradford assay. Load 20–40 µg total protein per lane.
2. Electrophoresis: Resolve on a 12–15% SDS-PAGE gel under reducing conditions. Transfer to PVDF or nitrocellulose membrane.
3. Blocking: Block for 1 hour at room temperature with 5% non-fat dry milk or 3% BSA in TBST.
4. Primary antibody incubation: Incubate with PRDX3 Mouse mAb (Ab349216) overnight at 4 °C at the manufacturer-recommended dilution (consult datasheet).
5. Secondary antibody: Incubate with HRP-conjugated anti-mouse IgG secondary antibody for 1 hour at room temperature.
6. Detection: Develop with ECL substrate. Expected band: ~28–30 kDa (monomer).

Immunofluorescence Protocol

1. Fixation: Fix cells in 4% paraformaldehyde for 15 minutes at room temperature. Permeabilize with 0.1–0.2% Triton X-100 for 10 minutes.
2. Blocking: Block with 3–5% normal goat serum or BSA for 1 hour.
3. Primary antibody: Apply PRDX3 Mouse mAb at manufacturer-recommended dilution overnight at 4 °C.
4. Secondary antibody: Incubate with fluorophore-conjugated anti-mouse IgG for 1 hour at room temperature, protected from light.
5. Counterstain and mount: Counterstain nuclei with DAPI; mount with anti-fade medium. Co-stain with a mitochondrial marker (e.g., MitoTracker or anti-TOMM20) to confirm co-localization.

All dilutions and incubation times should be optimized empirically and cross-referenced with the product datasheet.

Storage, Handling, and Quality Control

Storage conditions: Store PRDX3 Mouse mAb (Ab349216) at –20 °C upon receipt. For short-term use (up to 1 month), the antibody may be stored at 4 °C. Avoid repeated freeze-thaw cycles, as these can reduce antibody activity and specificity. Aliquot into single-use volumes before freezing if the antibody will be used intermittently.

Reconstitution: If supplied lyophilized, reconstitute in the volume of sterile water or PBS specified on the vial label and allow to equilibrate for 30 minutes before use. Consult the datasheet for carrier protein content and preservative information.

Quality control: Each lot of Aladdin Scientific antibodies is subject to quality control testing. Verify lot-specific data (SDS-PAGE purity, functional test results) on the Certificate of Analysis (CoA) available through your account portal or by contacting technical support.

Expiry: Use within the expiry date stated on the product label. Performance beyond the stated expiry cannot be guaranteed.

Troubleshooting Common Issues

Problem	Possible Cause	Suggested Action
No signal on Western blot	Antibody dilution too high; insufficient antigen; transfer failure	Titrate antibody; verify protein loading with housekeeping marker (e.g., β-actin); check transfer efficiency with Ponceau S stain
High background	Insufficient blocking; secondary antibody cross-reactivity; antibody concentration too high	Increase blocking time/concentration; increase wash stringency; reduce primary antibody concentration
Multiple non-specific bands	Protease degradation; sample overloading; cross-reactivity	Add fresh protease inhibitors; reduce protein load; confirm species reactivity on datasheet
Weak or absent IF signal	Over-fixation; inadequate permeabilization; wrong fixation method for epitope	Optimize fixation duration; test methanol fixation as alternative; increase antibody concentration
Diffuse mitochondrial staining pattern	Mitochondrial fragmentation due to cell stress	Optimize cell culture conditions; harvest at lower passage; co-stain with mitochondrial marker to confirm

Frequently asked questions

What is the expected molecular weight of PRDX3 detected by Western blot?

Under reducing SDS-PAGE conditions, PRDX3 migrates at approximately 28–30 kDa, corresponding to the monomeric form. Under non-reducing conditions, the disulfide-linked homodimer may appear at approximately 56–60 kDa. Always confirm the expected band position with the product datasheet, as post-translational modifications can shift apparent molecular weight.

Which species does PRDX3 Mouse mAb (Ab349216) recognize?

This antibody is raised against human PRDX3. Cross-reactivity with mouse, rat, or other species depends on epitope conservation and must be confirmed by consulting the product datasheet or Certificate of Analysis. Do not assume cross-reactivity without experimental validation.

Can PRDX3 Mouse mAb be used for immunoprecipitation (IP)?

Suitability for immunoprecipitation is not stated in the available source material for Ab349216. Contact Aladdin Scientific technical support or refer to the current product datasheet to confirm whether IP has been validated for this lot. In general, antibody performance in IP depends on epitope accessibility under native, non-denaturing conditions.

How should I handle the antibody to maximize its shelf life?

Store at –20 °C immediately upon receipt and avoid repeated freeze-thaw cycles by aliquoting into single-use volumes. For routine daily use, a working aliquot may be kept at 4 °C for up to one month. Keep the antibody away from light and do not vortex vigorously; gentle mixing by inversion or flicking is preferred.

What positive controls are recommended when using PRDX3 Mouse mAb?

Cell lines known to express high levels of PRDX3, such as HeLa, HEK293, or Jurkat cells, are commonly used as positive controls for PRDX3 detection. Recombinant PRDX3 protein can serve as a spike-in positive control for Western blot. Consult the product datasheet for lot-specific positive control recommendations.

References

1. Rhee SG, Chae HZ, Kim K. Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic Biol Med. 2005;38(12):1543-1552. doi:10.1016/j.freeradbiomed.2005.02.026
2. Cox AG, Winterbourn CC, Hampton MB. Mitochondrial peroxiredoxin involvement in antioxidant defence and redox signalling. Biochem J. 2010;425(2):313-325. doi:10.1042/BJ20091541
3. Bae YS, Oh H, Rhee SG, Yoo YD. Regulation of reactive oxygen species generation in cell signaling. Mol Cells. 2011;32(6):491-509. doi:10.1007/s10059-011-0276-3

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.