This Oxidative Stress Defense Western Blot Cocktail is designed to determine the relative abundance of several important proteins involved in the protection of cells against oxidative stress and the regulation of reactive oxygen species (ROS). Reactive oxygen species’ are produced naturally in cells as byproducts of the metabolism of oxygen as well as in response to various environmental stresses including UV radiation, pollutants, and heat exposure. Additionally, ROS levels can be altered by disease and injury, including cancer, neurodegenerative disease, cardiovascular disease, ischemia, stroke and aging. Reactive oxygen species also play an important role in cell signaling, a process called redox signaling. The regulation of ROS within cells is important for maintaining a proper homeostasis.

Superoxide dismutase 1 (SOD1) scavenges harmful superoxides (O2-) within cells protecting them from harmful oxidation of lipids, proteins and nucleic acids. Its altered expression levels have been linked to Down’s syndrome, ALS and various cancers. Similarly, the hydrogen peroxide(H2O2) scavenging enzyme, catalase, also regulated ROS concentrations within cells by reducing H202 into less reactive O2 and water. Thioredoxin is a small enzyme (12kDa) that facilitates the reduction of other enzymes via cysteine thiol-disulfide exchange. Thioredoxin is used by cells to reduce ROS amounts and in redox signaling processes. Finally, alpha smooth muscle actin was included in the cocktail as a loading control. Widely expressed, smooth muscle actin is involved in cell structure and motility.

These four readouts are easily resolved by western blot given their different molecular weights. Because they are all rabbit monoclonal antibodies, an anti-rabbit secondary should be used for detection.

Expected and observed MWs:

• Catalase: 60 kDa
• Smooth Muscle Actin: 42 kDa
• Superoxide Dismutase 1: 16 kDa
• Thioredoxin: 12 kDa

WB Notes:

• - WB samples should be heated to 95°C for 5 minutes in sample buffer before loading.
• - Suggested working concentration is 1X for primary antibody cocktail.
• - Suggested dilution buffer is 5% milk/PBS+0.05%Tween 20.

The cocktail contains 50% glycerol, can be stored at -20C. No aliquoting necessary.

• Kits/ Lysates/ Other
• Kits
• Antibody Cocktails
• Oxidative Stress