Manasa, C. and Bhandari, Rashna (2020) Back-Pyrophosphorylation Assay to Detect In Vivo InsP7-Dependent Protein Pyrophosphorylation in Mammalian Cells. Methods in Molecular Biology, 2091. pp. 93-105. ISSN 1064-3745

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Abstract

Protein pyrophosphorylation involves the transfer of a high-energy β-phosphate from inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (InsP7) to phosphorylated serine residues. Over a decade of research has established several proteins, involved in diverse physiological processes, as substrates of InsP7-mediated pyrophosphorylation. However, the need for detection of this posttranslational modification on endogenous proteins is paramount. “Back-pyrophosphorylation” is a simple technique to test whether a native protein undergoes InsP7-mediated pyrophosphorylation inside cells. The basis of this technique relies on the fact that a target protein isolated from cells with lower InsP7 levels exists in a hypo-pyrophosphorylated form as compared to the same protein isolated from cells with normal InsP7 levels. Hence, when radiolabeled InsP7 is added to a target protein immunoprecipitated from both these cell types, the hypopyrophosphorylated protein accepts a higher amount of radiolabeled phosphate when compared to the protein isolated from wild-type cells. This chapter provides detailed methods to identify an InsP7 target protein and conduct a back-pyrophosphorylation assay on a target protein immunoprecipitated from cells with normal versus reduced InsP7 levels, to confirm its endogenous pyrophosphorylation status.

Item Type:	Article
Subjects:	Molecular Biology
Depositing User:	Users 2 not found.
Date Deposited:	30 Nov 2019 06:43
Last Modified:	30 Nov 2019 06:43
URI:	http://cdfd.sciencecentral.in/id/eprint/933

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