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BMC Biochemistry

, 7:6

First Online: 28 February 2006Received: 30 September 2005Accepted: 28 February 2006

Abstract

BackgroundAvailable in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation for example, autoradiography with H-labeled anchor precursors are time consuming weeks-months, laborious and suffer from low sensitivity.

ResultsWe describe a new technique for detecting prenyl anchors in N-terminally glutathione S-transferase GST-labeled constructs of target proteins expressed in vitro in rabbit reticulocyte lysate and incubated with H-labeled anchor precursors. Alternatively, hemagglutinin HA-labeled constructs expressed in vivo in cell culture can be used. For registration of the radioactive marker, we propose to use a thin layer chromatography TLC analyzer. As a control, the protein yield is tested by Western blotting with anti-GST- or anti-HA- antibodies on the same membrane that has been previously used for TLC-scanning. These protocols have been tested with Rap2A, v-Ki-Ras2 and RhoA variant RhoA63L including the necessary controls. We show directly that RasD2 is a farnesylation target.

ConclusionSavings in time for experimentation and the higher sensitivity for detecting H-labeled lipid anchors recommend the TLC-scanning method with purified GST- or HA- tagged target proteins as the method of choice for analyzing their prenylation capabilities in vitro and in vivo and, possibly, also for studying the myristoyl and palmitoyl posttranslational modifications.

AbbreviationsFPPfarnesylpyrophosphate

FTasefarnesyltransferase

FTIfarnesyltransferase inhibitor

GAPGTPase activating protein

GEFguanine nucleotide exchange factor

GFPgreen fluorescent protein

GGPPgeranylgeranylpyrophosphate

GGTase1geranylgeranyltransferase 1

GGTase2geranylgeranyltransferase 2

GGTIgeranylgeranyltransferase inhibitor

GSTglutathione-S-transferase

HAhemagglutinin

PBSphosphate-buffered saline

PCRpolymerase chain reaction

PTasesprenyltransferases

PTMposttranslational modification

SDS-PAGEsodium dodecyl sulphate polyacrylamide gelelectrophoresis

TLCthin layer chromatography

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2091-7-6 contains supplementary material, which is available to authorized users.

Wolfgang Benetka, Manfred Koranda contributed equally to this work.

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Autor: Wolfgang Benetka - Manfred Koranda - Sebastian Maurer-Stroh - Fritz Pittner - Frank Eisenhaber

Fuente: https://link.springer.com/article/10.1186/1471-2091-7-6







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