Merck - S7150 - S7150 OxyBlot Protein Oxidation Detection Kit The OxyBlot Protein Oxidation Detection Kit provides the reagents to perform the immunoblot detection of carbonyl groups introduced into proteins

ITEM #: 11569435
O x idative modification of proteins by o x ygen 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 O x yBlot Kit provides reagents for simple and sensitive immunodetection of these carbonyl groups, which is a hallmark of the o x idation 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 mi x ture 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 20-188 /catalogue/item/20-188Reducing agent is very important for preventing o x idation: 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 o x idation of proteins that may occur after cell lysis. Lysis buffer containing either 1-2% 2-mercaptoethanol or 50 mM DTT sufficiently inhibits this o x idation, but has no adverse effect on the derivatization reaction in the O x yBlot protocol. Purified protein samples are also suitable for analysis with the O x yBlot Kit. For general information about protein e x traction, please check this link. /immunodetection/id3/proteine x traction
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S7150 OxyBlot Protein Oxidation Detection Kit The OxyBlot Protein Oxidation Detection Kit provides the reagents to perform the immunoblot detection of carbonyl groups introduced into proteins
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