| In Gel Kinase Assay
|
|
Author:
Date:
2017-03-05
[Abstract] Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol ‘In Gel Kinase Assay’, which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used ‘In Gel Kinase Assay’ protocol to show that the Arabidopsis thaliana sextuple mutant in the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR-ABA receptors; line pyr/pyl112458) is impaired in ABA-mediated activation of SnRK2.2, SnRK2.3 and OST1/SnRK2.6, as much as the triple mutant snrk2.2/2.3/2.6 (Gonzalez-Guzman et al., 2012).
[摘要] 正常发育和体内平衡需要细胞和组织中蛋白质磷酸化的适时时空调节。我们提出方案“凝胶激酶测定”，其可用于粗蛋白质提取物的蛋白激酶活性测量。我们已经成功地使用“凝胶激酶测定”方案来证明在ABA受体（PYR / PYL / RCAR-ABA受体）的PYRABACTIN RESISTANCE1 / PYR1-样/调节组分中的拟南芥线条pyr / pyl112458 ）在ABA介导的SnRK2.2，SnRK2.3和OST1 / SnRK2.6的活化中受损，多达三重突变体snrk2.2 / 2.3 / 2.6 （Gonzalez-Guzman等人，2012）。
背景 植物激素脱落酸（ABA）是涉及植物生长发育以及植物对非生物和生物胁迫的反应的关键信号。 ABA感知和信号通路由ABA受体（PYR / PYL / RCAR-ABA受体）的PYRABACTIN RESISTANCE1 / PYR1-调节组分，PP2C磷酸酶和SnRK2s激酶组成（在Antoni等人， ，，2011）。模块受体-ABA-磷酸酶通过调节ABA激活的SnRK2而以配体依赖的方式控制磷酸化信号级联。反过来，SnRK2s激酶使细胞核和细胞质中的无数效应物从转录因子（例如，ABFs）到离子通道（例如）磷酸化， ...
|
|
|
| Primer Extension Analysis Using AMV Reverse Transcriptase
|
|
Author:
Date:
2012-07-20
[Abstract] Primer extension analysis is a useful method to determine the transcription start point or a processing site on an RNA molecule. It can also allow a quantitative measurement of an RNA species.
[摘要] 引物延伸分析是确定RNA分子上的转录起始点或加工位点的有用方法。 它还可以允许RNA物质的定量测量。
|
|
|
| Synthesis of 5’ end-labeled RNA
|
|
Author:
Date:
2012-07-20
[Abstract] 5’ end-labeled RNA molecules are useful substrates to analyse the endo- and exonucleolytic activities of various ribonucleases. Here two protocols are given to synthesize P32 labeled RNAs with a 5’ PPP or 5’ P moiety. 5’ exoribonucleases generally do not work on 5’ PPP RNA and require a 5’ P substrate. The activity of certain endoribonucleases like Escherichia coli (E. coli) RNase E or Bacillus subtilis (B. subtilis) RNase Y can be stimulated by a 5’ P moiety.
[摘要] 5'末端标记的RNA分子是用于分析各种核糖核酸酶的内切和外切核酸活性的有用底物。 这里给出两个方案来合成具有5'PP或5'P部分的P sup 32标记的RNA。 5'外核糖核酸酶通常不在5'PPP RNA上起作用并且需要5'P底物。 某些内切核糖核酸酶如大肠杆菌（大肠杆菌）核糖核酸酶E或枯草芽孢杆菌（枯草芽孢杆菌 ）核糖核酸酶Y可以被5'P部分刺激
|
|
|