Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo

BMC Biochemistry
Wolfgang BenetkaFrank Eisenhaber

Abstract

Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with 3H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity. We 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 3H-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. Savings in time for experimentation and the higher sensitivity for detecting 3H-labeled lipid anchors recommend the TLC-scanning method with purified GST- (or HA-) ...Continue Reading

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Citations

Jul 27, 2011·Analytical and Bioanalytical Chemistry·Gudrun Nürenberg, Dietrich A Volmer
Mar 6, 2009·Proceedings of the National Academy of Sciences of the United States of America·Zhihua LiuPeter T Lansbury
Mar 11, 2009·Molecular and Cellular Biology·Bas PonsioenKees Jalink
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Apr 7, 2007·PLoS Computational Biology·Sebastian Maurer-StrohFrank Eisenhaber
Sep 9, 2017·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Ekaterina S MarakasovaAncha Baranova
Feb 17, 2009·Nature Chemical Biology·Uyen T T NguyenKirill Alexandrov
May 1, 2010·Chembiochem : a European Journal of Chemical Biology·Uyen T T NguyenKirill Alexandrov
Sep 22, 2010·Journal of Neuroscience Research·Inge SmoldersMarcel Ameloot
Feb 20, 2018·Critical Reviews in Biochemistry and Molecular Biology·Saeideh Nakhaei-RadMohammad Reza Ahmadian
Jun 19, 2019·Nature Structural & Molecular Biology·Shafi KuchayMichele Pagano
Jan 24, 2015·Analytical Chemistry·Ruchika P BhawalSaiful M Chowdhury

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Datasets Mentioned

BETA
BC070031
BC013419
BC013572

Methods Mentioned

BETA
GTPases
electrophoresis
geranylgeranylation
electrophoretic shift
fluorescence
transfection
fluorescence microscopy
immunoprecipitation
PCR
FCS

Software Mentioned

Metamorph

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