Methionine Sulfoxide Western Blot, BioAssay(TM) Kit

Several amino acid residues on proteins are modified upon reaction with reactive oxygen species (ROS). Although it has been extensively documented that methionine residues can be oxidized to methionine sulfoxide and, in contrast to some other adducts produced upon oxidation, methionine sulfoxide residues can be converted back to methionine by the action of methionine sulfoxide reductase. Thus, in addition to methionine sulfoxide residues serving as biomarkers for protein oxidation, this particular oxidation may play a role in redox signaling within the body.\n\nIntended Use:\nThis kit has been tested for Western Blot. Other uses have not been tested.\n\nKit Components:\nM3022-01A: Methionine Sulfoxide Pab Rabbit 1x100ul\nM3022-01B: Standard protein with extensive methionine sulfoxide modifications, 1x100ul\nM3022-01C: Methionine Sulfoxide 2mM solution used to suppress immunoreactivity, 1x100ul\n\nStorage and Stability:\nStore kit and its components at -20 degrees C until use. Aliquot to avoid repeated freeze/thaw cycles. For maximum recovery of product, centrifuge the original vial prior to removing the cap. \n\nReagent Preparation:\n1. M3022-01A: Methionine Sulfoxide Pab Rabbit . The recommended starting dilution for Western Blot is 1:2000. \n2. M3022-01B: Standard: Load 10ul/lane. The standard is a protein with extensive methionine sulfoxide modifications.\n3. M3022-01C: Methionine Sulfoxide: 2mM solution of free methionine sulfoxide that can be used to ensure specificity and to confirm reactivity by blocking all specific reactivity of the antibody. Incubate the antibody with the 2mM solution of methionine sulfoxide for one hour at 37 degrees C then overnight at 4 degrees C.\n\nNotes for Western Blot Procedure:\nThe antibody and standard are compatible with common Western Blot procedures. For best results, the following is suggested:\n1. The nitrocellulose membrane should be blocked with PBS, 5% dry milk over night.\n2. The antibody should be diluted 1:2000 in PBS, 1% dry milk, 0.05% tween-20.\n3. Purified proteins being tested as targets for methionine oxidation should be treated with and without 200mM H2O2 overnight at RT. These samples should then be run on 4-20% SDS gel followed by common Western Blot procedures.\n4. Non-purified proteins from cells or tissue extracts should not be treated with H2O2, but protein enrichment may be required.

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