Oxidative modification of proteins by oxygen free radicals and other reactive species occurs in physiologic and pathologic processes. As a consequence of the modification, carbonyl groups are introduced into protein side chains by a site-specific mechanism. The OxyBlot Kit provides reagents for simple and sensitive immunodetection of these carbonyl groups, which is a hallmark of the oxidation status of proteins.
Application: Preparation of Cell Lysate 1. Wash adherent cells twice in the dish or flask with ice-cold PBS and drain off PBS. Wash non-adherent cells in PBS and centrifuge at 800 to 1000 rpm in a table-top centrifuge for 5 minutes to pellet the cells. 2. Add ice-cold RIPA buffer to cells (1 ml per 107 cells/100 mm dish/150 cm2 flask; 0.5 ml per 5 x 106 cells/60 mm dish/75 cm2 flask). 3. Scrape adherent cells off the dish or flask with either a rubber policeman or a plastic cell scraper that has been cooled in ice-cold distilled water. Transfer the cell suspension into a centrifuge tube. Gently rock the suspension on either a rocker or an orbital shaker in the cold room for 15 minutes to lyse cells. 4. Centrifuge the lysate at 14,000 x g in a precooled centrifuge for 15 minutes. Immediately transfer the supernatant to a fresh centrifuge tube and discard the pellet. 5. Dilute the cell lysate at least 1 : 10 before determining the protein concentration because of the interference of the detergents in the lysis buffer with the Coomassie-based reagent. At this step, the sample can be divided into aliquots and stored at -20¡C for up to a month. TIP: When working with large volumes of non-adherent cells, the cells may not be cooled quickly enough to maintain the activity of the protein being studied. In this case, pour the cell suspension into a mixture of an equal mass of 2 x PBS and ice, then collect the cells by centrifugation and perform the lysis as described above. RIPA lysis buffer: Catalog number 20-188http://www.millipore.com/catalogue/item/20-188Reducing agent is very important for preventing oxidation: The protein lysate may be prepared using a variety of protocols. Most standard buffers such as RIPA and Triton are also suitable. It is recommended that a reducing agent be added to the lysis buffer to prevent the oxidation of proteins that may occur after cell lysis. Lysis buffer containing either 1-2% 2-mercaptoethanol or 50 mM DTT sufficiently inhibits this oxidation, but has no adverse effect on the derivatization reaction in the OxyBlot protocol. Purified protein samples are also suitable for analysis with the OxyBlot Kit. For general information about protein extraction, please check this link. http://www.millipore.com/immunodetection/id3/proteinextraction
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