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Available in vitro and in vivo methods for verifying protein substrates for …


Biology Articles » Biochemistry » Protein Biochemistry » Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo

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

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

Wolfgang Benetka* 1, Manfred Koranda* 1, Sebastian Maurer-Stroh1,2, Fritz Pittner3 and Frank Eisenhaber1

1Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, A-1030 Vienna, Austria
2VIB – SWITCH lab, Pleinlaan 2, B-1050 Brussels, Belgium
3University Vienna, Department of Biochemistry, Dr.-Bohr-Gasse 9, A-1030 Vienna, Austria

 

Background

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.

Results

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.

Conclusion

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-) 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.

BMC Biochemistry 2006, 7:6. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.


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