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 not tested. Recommended Dilutions: Immunohistochemistry (paraffin): Pretreat tissues using a citrate buffer, pH 6.0 and high heat epitope retrieval techniques. 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: Store product at 4C if to be used immediately within two weeks. For long-term storage, aliquot to avoid repeated freezing and thawing and store at -20C. Aliquots are stable at -20C for 12 months after receipt. Dilute required amount only prior to immediate use. Further dilutions can be made in assay buffer. Caution: FITC conjugates are sensitive to light. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Note: Applications are based on unconjugated antibody.
Purity:
Purified by Protein A affinity chromatography.
Form:
Supplied as a liquid in PBS, pH 7.2. No preservative added. Labeled with Fluorescein isothiocyanate (FITC).
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