Rho Kinase(ROCK)活性測定キット 96-Well ROCK Activity Assay Kit

ROCK(Rho-associated kinase)でMYPT1(myosin phosphatase target subunit 1)をThr696の特異的リン酸化することで、ROCK活性を検出するキットです。ポジティブコントロールとして組換え体活性型ROCK-Ⅱが含まれています。

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組換え体MYPT1をコートしたプレートにROCKを含む細胞／組織ライセートを添加してインキュベートした後、抗リン酸化MYPT1(Thr696)抗体でリン酸化MYPT1を検出します。

Figure 1: ROCK-II Activity Assay
Left: Active ROCK-II was incubated for 60 minutes at 30°C. Right: 2.5 ng of active ROCK-II was incubated at 30°C for times as shown. Phosphorylation of MYPT1 substrate was detected by anti-phospho-MYPT1 (Thr696) antibody as described in Assay Protocol.

Figure 2: ROCK-II Activity Immunoblot Assay
25 μL of 1X Kinase Buffer containing 10 ng of active ROCK-II was incubated with 50 μL of 1X Kinase Buffer containing 0.2 mM ATP and 500 ng of recombinant MYPT1 for 30 minutes at 30°C. Kinase reaction was stopped by adding 25 μL of 4X SDS-PAGE Sample Buffer. Lane 1, 3, 5, 7: Without kinase (negative control); Lane 2, 4, 6, 8: with kinase. 200 ng (Lane 1 and 2), 100 ng (Lane 3 and 4), 50 ng (Lane 5 and 6) or 25 ng (Lane 7 and 8) of recombinant MYPT1 substrate were loaded onto SDS-PAGE. Phosphorylation of MYPT1 substrate was detected by anti-phospho-MYPT1 (Thr696) antibody as described in the Assay Protocol for Cat. # STA-415.