Multiwell filtration assay for rapid determination of protein phosphatase activity.
Multiwell filtration assay for rapid determination of protein phosphatase activity.
复制标题
用于快速测定蛋白磷酸酶活性的多孔过滤测定。
DOI：
10.1006/abio.1993.1331
复制
发表时间：
1993
影响因子：
2.9
通讯作者：
Chen,ZH
中科院分区：
文献类型：
--
作者：
Gopalakrishna,R;Gundimeda,U;Wilson,JC;Chen,ZH
和TIPS，以及在各种亚细胞组分和层析组分中没有金属、抑制剂或激活剂(1，2)。因此，需要一种能够方便、快速地测定大量样品中PP活性的方法。在传统的方法中，PP测定是在试管或微型福格试管中进行的，使用“P标记的蛋白质底物(3-5)，用三氯乙酸(TCA)沉淀和离心法(3-5)除去磷蛋白后，PP反应释放的P被计数。在可能受到磷化蛋白底物蛋白分解干扰的情况下，释放的P_1转化为磷钼酸络合物，并用苯和异丁醇(4)等有机溶剂进行萃取。这些成熟的传统方法使用试管或微隔离管是有效的，适用于有限数量的样品中PP的测定。最近，我们报道了一种用多孔板和合适的滤片测定蛋白激酶C的方法(6)。这种方法的独特之处在于，测定蛋白激酶C活性所需的孵育和过滤都在同一多孔板中进行，不需要转移样品。由于多孔滤板也适用于滤液的定量收集，现在我们应用这种多孔超滤方法将PP反应释放的P从酸沉淀的P标记蛋白中分离出来。这种多孔过滤方法有助于在大量样品中快速测定PP活性。从MilliPore获得了多筛系统96孔板和真空歧管，板上装有过滤器(Durapore膜，孔径0.45pum)和真空歧管。组蛋白H1被蛋白激酶C(7)磷酸化，每毫克组蛋白H1约掺入20~25nmol的“P”。在建立分析方法之前，“用缓冲液A(50 mM Tris-HCl，pH 7.5/1 mM EDTA/1 mM DDT/0.1M氯化钠/牛血清白蛋白(0.2 mg/ml)/1pum胃抑素A)稀释P-标记组蛋白H1样品，使底物反应混合物达到每50pl 75 pmol”P-标记组蛋白H1(基于掺入的“P”)。
& TIPS and absence of metals, inhibitors, or activators in various subcellular fractions and chromatographic frac-tions (1, 2). Therefore, there is a need for methods which can conveniently and rapidly determine PP activities in larger numbers of samples. In the conventional approach PP assays are carried out in test tubes or micro-fuge tubes by using" P-labeled protein substrates (3–5). The" P, liberated by the PP reaction is counted after removing the phosphoprotein by trichloroacetic acid (TCA) precipitation and centrifugation (3–5). In the cases where the interference by proteolysis of phospho-protein substrate is expected, the liberated “P, is con-verted into a phosphomolybdate complex, and the complex is extracted with organic solvents such as benzene and isobutanol (4). These well-established conventional methods which use test tubes or microfuge tubes are efficient and suitable for PP determinations in a limited number of samples. Recently we have reported a protein kinase C assay using multiwell plates with fitted filtration discs (6). The unique aspect of this method is that both the incubations and the filtrations necessary for the determination of protein kinase C activity are carried out in the same multiwell plate without the need for the transfer of the sample. Since the multivvell filtra-tion plates are also suited for the quantitative collection of the filtrate, now we have applied this multiwell ultra-filtration approach to separate the “P, released by the PP reaction from the acid-precipitated" P-labeled pro-teins. This multiwell filtration approach facilitated rapid determinations of PP activity in larger numbers of samples.The MultiScreen system 96-well plates with fitted filters (Durapore membrane, pore size 0.45 pum) and vacuum manifold for the multivvell plates were obtained from Millipore. Histone H1 was phosphorylated with protein kinase C (7), and approximately 20 to 25 nmol of “P was incorporated per milligram histone H1. Before setting up the assay,“P-labeled histone H1 sample was diluted with buffer A (50 mM Tris–HCl, pH 7.5/1 mM EDTA/1 mM DDT/0.1 M NaCl/bovine serum albumin (0.2 mg/ml)/1 puM leupeptin./0.3 puM pepstatin A) to bring the substrate reaction mixture to 75 pmol of" P-labeled histone H1 (based on the incorporated “P) per 50 pl.
登录
查看更多内容
心脏肌浆网囊泡功能异质性的生化证据。
影响因子：
4.8
作者：
L. Jones;S. Cala
通讯作者：
S. Cala
心脏肌浆网钙通道的单通道和 45Ca2 通量测量。
影响因子：
3.4
作者：
Rousseau,E;Smith,JS;Henderson,JS;Meissner,G
通讯作者：
Meissner,G
钙通道测定。
影响因子：
--
作者：
H. Glossmann;D. Ferry
通讯作者：
D. Ferry
DOI：
10.1016/s0021-9258(18)61565-0
发表时间：
1987-02
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
F. Lattanzio;Robert;Schlattererl;Michael Nicart;Kevin;Campbell;John L. SutkoSQII
通讯作者：
F. Lattanzio;Robert;Schlattererl;Michael Nicart;Kevin;Campbell;John L. SutkoSQII
揭示阿拉甲辛对心脏肌膜囊泡的 (Na ,K )-ATP 酶、β-肾上腺素受体偶联腺苷酸环化酶和 cAMP 依赖性蛋白激酶活性的影响。
影响因子：
4.8
作者：
L. Jones;S. Maddock;H. R. Besch
通讯作者：
H. R. Besch