The cellular production of highly reactive nitrogen species derived from nitric oxide, such as peroxynitrite, nitrogen dioxide and nitryl chloride, leads to the nitration of tyrosine resides in tissue proteins. The extent of protein nitrotyrosine formation provides an index of the production of reactive nitrogen species and potential cell damage over a period of time. Nitrotyrosine can be measured by amino-acid analysis of protein hydrolysates and detected, estimated semi-quantitatively and located in cells and tissues by immunocytochemical techniques using antibodies directed against the nitrotyrosine hapten. Applications: Suitable for use in Western Blot and Immunohistochemistry. Other applications have not been tested. Recommended Dilutions: Western Blot: 1:100-1:1000 when tested against nitrated BSA and nitrated ovalbumin. Immunohistochemistry (Paraffin): 1:500 using mouse brain (animal treated with Zymosan) with pretreatment with citrate buffer, pH 6.0. Optimal dilutions to be determined by the researcher. Positive Control: Tyrosine residues can be nitrated directly in situ on tissue sections using peroxynitrite to create a positive control. After the tissue section is deparaffinized, the slide is covered with a drop of PBS, pH 7.4 into which 10ul of peroxynitrite solution are mixed. Care should be taken that the solution maintains a pH of 7.4. Storage and Stability: May be stored at 4C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20C. Aliquots are stable for 12 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Purity:
Purified by Protein A affinity chromatography.
Form:
Supplied as a liquid in PBS, pH 7.6, 0.09% sodium azide.
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