Western blot analysis was performed on membrane enriched extracts (30 ug lysate) of HeLa (Lane 1), MCF7 (Lane 2), HCT 116 (Lane 3), A549 (Lane 4), A-431 (Lane 5) and Hep G2 (Lane 6). The blot was probed with 2-Cys Peroxiredoxin Mouse monoclonal Antibody (Product# LF-MA0073, 2 ug/ml) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 ug/ml, 1:4000 dilution). A 22 kDa band corresponding to 2-Cys Peroxiredoxin was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Human|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Purified, recombinant, human 2-Cys peroxiredoxin protein expressed in E. coli.|
|Purification||Ammonium sulfate precipitation|
|Storage buffer||HEPES with 0.15M NaCl, 0.01% BSA, 50% glycerol|
|Contains||0.03% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|ELISA (ELISA)||Assay dependent|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Western Blot (WB)||1-3 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Western Blot (WB)||See 1 publications below|
A suggested positive control for this product is HeLa cells.
Peroxiredoxin (Prx) is a growing peroxidase family, whose mammalian members have been known to connect with cell proliferation, differentiation, and apoptosis. Many isoforms (about 50 proteins), collected in accordance to the amino acid sequence homology, particularly amino-terminal region containing active site cysteine residue, and the thiol-specific antioxidant activity, distribute throughout all the kingdoms. Among them, mammalian Prx consists of 6 different members grouped into typical 2-Cys, atypical 2-Cys Prx, and 1-Cys Prx. Except Prx VI belonging to 1-Cys Prx subgroup, the other five 2-Cys Prx isotypes have the thioredoxin-dependent peroxidase (TPx) activity utilizing thioredoxin, thioredoxin reductase, and NADPH as a reducing system. Mammalian Prxs are 20-30 kilodalton in molecular size and vary in subcellular localization: Prx I, II, and VI in cytosol, Prx III in mitochondria, Prx IV in ER and secretion, Prx V showing complicated distribution including peroxisome, mitochondria and cytosol.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Treating gout with pegloticase, a PEGylated urate oxidase, provides insight into the importance of uric acid as an antioxidant in vivo.
LF-MA0073 was used in western blot to investigate the function of uric acid as an antioxidant in vivo
|Hershfield MS,Roberts LJ,Ganson NJ,Kelly SJ,Santisteban I,Scarlett E,Jaggers D,Sundy JS||Proceedings of the National Academy of Sciences of the United States of America (107:14351)||2010|