Cyclooxygenases: Methods and Protocols by Roderick J. Flower (auth.), Samir S. Ayoub, Roderick J.

By Roderick J. Flower (auth.), Samir S. Ayoub, Roderick J. Flower, Michael P. Seed (eds.)

Since the invention of the pharmacological and toxicological value of inhibiting the cyclooxygenase (COX) enzymes by means of non-steroidal anti inflammatory medicinal drugs (NSAIDs), a lot study has long gone into the advance of tips on how to research the organic services of COX-1 and COX-2. In Cyclooxygenases: tools and Protocols, specialists and pioneers within the box current the main updated in vitro and in vivo recommendations in many instances utilized in COX study. the amount delves into crucial themes equivalent to the purification, cloning, and expression of COX enzymes in addition to in vitro assays geared toward opting for the inhibitory efficiency of gear on COX-1 and COX-2 actions, with chapters describing protocols used for the extraction and dimension of the prostanoids. This quantity additionally describes in vivo ailment versions used to check the jobs of COX-1 and COX-2 in gastrointestinal damage, irritation, and ache. As a e-book within the hugely winning Methods in Molecular Biology™ sequence, the protocols chapters comprise short introductions to their respective issues, lists of the required fabrics and reagents, step by step, effectively reproducible laboratory protocols, and notes on troubleshooting and fending off identified pitfalls.

Authoritative and state of the art, Cyclooxygenases: tools and Protocols serves as an quintessential device for all scientists looking the therapy of irritation, discomfort, fever, and different destructive conditions.

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As such, COX enzymes represent important pharmacological targets in the treatment of pain, inflammation, and fever. However, due to the typically low expression level of COX enzymes in most primary tissues, large amounts of highly purified enzyme for in depth study is difficult to obtain. Overexpression of COX in the baculovirus expression system represents a quick and efficient way to overcome this problem and obtain large amounts of catalytically active enzyme. Here, we present a step-by-step approach for COX expression in the baculovirus system.

Add 1 ml 70% EtOH (pre-chilled to −20°C), and centrifuge RNA for 10 min at 12,000 × g at 4°C. Carefully remove the supernatant as described above (steps 4 and 5). Allow to air-dry 10 min at RT. 25 ml nuclease-free water by pipetting up and down five times. 25 ml 2× Binding Solution and mix thoroughly. Add RNA sample to 1 tube Oligo(dT) Cellulose, and mix well by inversion. Heat the mixture for 5 min at 65°C. Place tube on a platform shaker, and rock gently for 60 min at RT. Centrifuge at 3,000 × g for 3 min at RT, and aspirate to remove supernatant.

Harvest as before, and use this second harvest to generate larger virus stocks. 3. Seed 250 ml Sf 9 cells at 1 × 106/ml in a disposable Erlenmeyer shake flask in Sf-900 II medium containing 5% FBS, for each virus. 1 containing the desired recombinant viruses to the cells. Monitor viability and cell diameter by Cedex. Cells should stop growing after ~24 h, and diameters should increase at least 4–5 mm (see Note 3). Harvest medium when the average cell diameter has increased and the viability is between ~50% and 70%; store at 4ºC.

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