Eppendorf GELoader^®, Eppendorf Quality^TM, 0.5 – 20 µL

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Catalog#: 022351656

In vitro Assays to Evaluate Specificity and Affinity in Protein-phospholipid Interactions

Hendry Susila, Snježana Jurić, Katarzyna Gawarecka, Kyung Sook Chung, Suhyun Jin, Soo-Jin Kim, Zeeshan Nasim, Geummin Youn and Ji Hoon Ahn

Published online: 2022-05-20

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Phosphatase Protection Assay: 14-3-3 Binding Protects the Phosphate group of RSG from λ Protein Phosphatase

Author:

Takeshi Ito and Yohsuke Takahashi,

Date:

2015-02-05

[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。

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Eppendorf GELoader®, Eppendorf QualityTM, 0.5 – 20 µL

Eppendorf GELoader®, Eppendorf QualityTM, 0.5 – 20 µL

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Eppendorf GELoader^®, Eppendorf Quality^TM, 0.5 – 20 µL

Eppendorf GELoader^®, Eppendorf Quality^TM, 0.5 – 20 µL