Difference between revisions of "SPINE"
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=='''A detailed protocol to detect the interaction between [[RocG]] and [[GltC]]:''' == | =='''A detailed protocol to detect the interaction between [[RocG]] and [[GltC]]:''' == | ||
Revision as of 13:15, 21 December 2012
SPINE is a method to detect in vivo protein-protein interactions PubMed
Contents
See the principle
A detailed protocol to detect the interaction between RocG and GltC:
1 litre of a B. subtilis culture was grown to an OD600 of approx. 1.0 and incubated with 0.6% formaldehyde ( 4% stock solution in PBS, pH 6.5!) for 20 minutes @ 37°C on a shaker. The cells were harvested and washed once in 1 X PBS pH 6.5. The pellets can then be stored @ -20 °C. The GltC protein was expressed carrying a Strep-tag and RocG expression was induced by arginine (PubMed). Expression of the Strep-tagged GltC protein allows to test the functionality of the protein. Crude extracts (10-15 ml) were prepared by using a French Press. After a centrifugation step for 1 h @ 27.000 g the clarified crude extracts were loaded onto a Streptactin sepharose column (0.5-1 ml matrix) to isolate the cross-linked protein complexes (the detailed procedure for protein purification is described in the IBA manual, http://www.iba-go.com/). After the purification of the protein complexes the crosslinks can be resolved by boiling the samples in Laemmli buffer for 10-15 minutes @ 95 °C (PubMed). A 12.5% SDS gel was loaded with the samples and the proteins were then visualized by silver-staining. The interaction partner/s were identified by mass spectroscopy and Western blotting.
Preparation of the formaldehyde stock solution (max. 4% in 1 X PBS pH 6.5): We use para-formaldehyde (a white powder). para-formaldehyde dissolves within approx. 20-30 minutes in 1 X PBS for @ 65 to 70 °C.
The sepharose matrix was purchased from the IBA company, Göttingen (http://www.iba-go.com/).
Relevant plasmids:
for use in B. subtilis (multicopy plasmids): pGP380, pGP382
for use in B. subtilis (chromosomal integration under the control of the native promoter): pGP1389
for use in E. coli: pGP172, pGP574
Biotin-containing proteins that are purified with the Strep-Tactin column
The reference for the method:
Christina Herzberg, Lope Andrés Flórez Weidinger, Bastian Dörrbecker, Sebastian Hübner, Jörg Stülke, Fabian M Commichau
SPINE: a method for the rapid detection and analysis of protein-protein interactions in vivo.
Proteomics: 2007, 7(22);4032-5
[PubMed:17994626]
[WorldCat.org]
[DOI]
(P p)
SPINE for membrane proteins:
Volker S Müller, Peter R Jungblut, Thomas F Meyer, Sabine Hunke
Membrane-SPINE: an improved method to identify protein-protein interaction partners of membrane proteins in vivo.
Proteomics: 2011, 11(10);2124-8
[PubMed:21472855]
[WorldCat.org]
[DOI]
(I p)
Other studies that made use of SPINE
Additional references: PubMed
Felix M P Mehne, Katrin Gunka, Hinnerk Eilers, Christina Herzberg, Volkhard Kaever, Jörg Stülke
Cyclic di-AMP homeostasis in bacillus subtilis: both lack and high level accumulation of the nucleotide are detrimental for cell growth.
J Biol Chem: 2013, 288(3);2004-17
[PubMed:23192352]
[WorldCat.org]
[DOI]
(I p)
Frederik M Meyer, Matthieu Jules, Felix M P Mehne, Dominique Le Coq, Jens J Landmann, Boris Görke, Stéphane Aymerich, Jörg Stülke
Malate-mediated carbon catabolite repression in Bacillus subtilis involves the HPrK/CcpA pathway.
J Bacteriol: 2011, 193(24);6939-49
[PubMed:22001508]
[WorldCat.org]
[DOI]
(I p)
Jens J Landmann, Ricarda A Busse, Jan-Hendrik Latz, Kalpana D Singh, Jörg Stülke, Boris Görke
Crh, the paralogue of the phosphocarrier protein HPr, controls the methylglyoxal bypass of glycolysis in Bacillus subtilis.
Mol Microbiol: 2011, 82(3);770-87
[PubMed:21992469]
[WorldCat.org]
[DOI]
(I p)
Martin Lehnik-Habrink, Joseph Newman, Fabian M Rothe, Alexandra S Solovyova, Cecilia Rodrigues, Christina Herzberg, Fabian M Commichau, Richard J Lewis, Jörg Stülke
RNase Y in Bacillus subtilis: a Natively disordered protein that is the functional equivalent of RNase E from Escherichia coli.
J Bacteriol: 2011, 193(19);5431-41
[PubMed:21803996]
[WorldCat.org]
[DOI]
(I p)
A K W Elsholz, K Hempel, S Michalik, K Gronau, D Becher, M Hecker, U Gerth
Activity control of the ClpC adaptor McsB in Bacillus subtilis.
J Bacteriol: 2011, 193(15);3887-93
[PubMed:21622759]
[WorldCat.org]
[DOI]
(I p)
Frederik M Meyer, Jan Gerwig, Elke Hammer, Christina Herzberg, Fabian M Commichau, Uwe Völker, Jörg Stülke
Physical interactions between tricarboxylic acid cycle enzymes in Bacillus subtilis: evidence for a metabolon.
Metab Eng: 2011, 13(1);18-27
[PubMed:20933603]
[WorldCat.org]
[DOI]
(I p)
Martin Lehnik-Habrink, Henrike Pförtner, Leonie Rempeters, Nico Pietack, Christina Herzberg, Jörg Stülke
The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complex.
Mol Microbiol: 2010, 77(4);958-71
[PubMed:20572937]
[WorldCat.org]
[DOI]
(I p)
Fabian M Commichau, Fabian M Rothe, Christina Herzberg, Eva Wagner, Daniel Hellwig, Martin Lehnik-Habrink, Elke Hammer, Uwe Völker, Jörg Stülke
Novel activities of glycolytic enzymes in Bacillus subtilis: interactions with essential proteins involved in mRNA processing.
Mol Cell Proteomics: 2009, 8(6);1350-60
[PubMed:19193632]
[WorldCat.org]
[DOI]
(I p)
Fabian M Commichau, Christina Herzberg, Philipp Tripal, Oliver Valerius, Jörg Stülke
A regulatory protein-protein interaction governs glutamate biosynthesis in Bacillus subtilis: the glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC.
Mol Microbiol: 2007, 65(3);642-54
[PubMed:17608797]
[WorldCat.org]
[DOI]
(P p)
