{{'Search' | translate}}

Amylose Resin


Company: New England Biolabs
Catalog#: E8021
Other protocol()

Phosphatase Protection Assay: 14-3-3 Binding Protects the Phosphate group of RSG from λ Protein Phosphatase
[Abstract]  14-3-3 proteins regulate diverse cellular processes in eukaryotes by binding to phospho-serine or threonine of target proteins. One of the physiological functions of 14-3-3 is to bind and protect phosphate groups of the target proteins against phosphatases. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in the feedback regulation of biosynthetic genes of plant hormone gibberellin. 14-3-3 binds to phospho-Ser-114 in RSG. Ca2+-dependent protein kinase NtCDPK1 was identified as a kinase that phosphorylates Ser-114 of RSG. Our recent study revealed that NtCDPK1 forms a heterotrimer with RSG and 14-3-3 and that 14-3-3 was transferred from NtCDPK1 to phosphorylated RSG (Ito et al., 2014). In the course of the ... [摘要]  14-3-3蛋白通过结合靶蛋白的磷酸 - 丝氨酸或苏氨酸来调节真核生物中的多种细胞过程。 14-3-3的生理功能之一是结合和保护靶蛋白的磷酸基团免受磷酸酶。生长生长的表达(RSG)是涉及植物激素赤霉素的生物合成基因的反馈调节的烟草(烟草属)转录因子。 14-3-3结合RSG中的磷酸-Ser-114。 Ca 2+ - 依赖性蛋白激酶NtCDPK1被鉴定为磷酸化RSG的Ser-114的激酶。我们最近的研究揭示NtCDPK1与RSG和14-3-3形成异源三聚体,并且14-3-3从NtCDPK1转移到磷酸化RSG(Ito等人,2014)。在研究过程中,我们发现14-3-3在体外保护RSG的磷酸基团免受λ蛋白磷酸酶的影响。在这里,我们描述了体外磷酸酶保护测定的协议。为了检测蛋白质的磷酸化状态,我们使用Phos-tag SDS-PAGE和放射自显影。该方案可以适用于磷酸蛋白结合蛋白是否保护靶蛋白的磷酸基团免于磷酸酶的检查,尽管蛋白激酶可能是靶蛋白磷酸化所需的。

In vitro Protein Ubiquitination Assays
[Abstract]  Ubiquitin can be added to substrate protein as a protein tag by the concerted actions of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3). At the present of E1 and ubiquitin, E2 activity can be determined by the thio-ester formation. The E3 activity of a putative protein as well as the E2/E3 or E3/substrate specificities also can be explored by in vitro ubiquitination assay. The result can be detected by western blot with certain antibody. Purified proteins expressed from bacterial system are always used in this assay. [摘要]  泛素可以通过泛素激活酶(E1),泛素缀合酶(E2)和泛素蛋白连接酶(E3)的协同作用作为蛋白标签添加到底物蛋白中。 在E1和泛素的存在下,E2活性可以通过硫酯形成来确定。 推定的蛋白质的E3活性以及E2/E3或E3 /底物特异性也可以通过体外泛素化测定来研究。 结果可以通过用某些抗体的western印迹来检测。 在该测定中总是使用从细菌系统表达的纯化蛋白质。