Difference between revisions of "SPINE"
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| − | SPINE is a methhod to detect in vivo protein-protein interactions | + | SPINE is a methhod to detect in vivo protein-protein interactions [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=+17994626 PubMed] |
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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: | ||
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The sepharose matrix was purchased from the IBA company, Göttingen (http://www.iba-go.com/). | The sepharose matrix was purchased from the IBA company, Göttingen (http://www.iba-go.com/). | ||
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| + | The refereence for the method: | ||
| + | Herzberg, C., Flórez Weidinger, L. A., Dörrbecker, B., Hübner, S., Stülke, J. & Commichau, F. M. (2007) SPINE: A method for the rapid detection and analysis of protein-protein interactions in vivo. ''Proteomics'' 7: 4032-4035. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=+17994626 PubMed] | ||
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| + | Other studies that made use of SPINE | ||
| + | # Commichau, F. M., Herzberg, C., Tripal, P., Valerius, O., and Stülke, J. (2007) A regulatory protein-protein interaction governs glutamate biosynthesis in ''Bacillus subtilis'': The glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC. Mol Microbiol 65: 642-654. [http://www.ncbi.nlm.nih.gov/sites/entrez/17608797 PubMed] | ||
Revision as of 17:26, 17 April 2009
SPINE is a methhod to detect in vivo protein-protein interactions PubMed
A detailed protocol to detect the interaction between RocG and GltC:
1 litre of a Bacillus subtilis culture was grown to an OD600 of approx. 1.0 and incubated with 0.6% formaldehyde (stock solution 4% 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 (see Commichau et al., 2007 Mol Microbiol). 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 (Herzberg et al., 2007 Proteomics). A 12.5% SDS gel was loaded with the samples and the proteins were then visualized by silver-staining. We identified the interaction partner/s by mass spectroscopy and Western blotting.
Preparation of the formaldehyde stock solution (max. 4% in 1X PBS pH 6.5): We use paraformaldehyde (a white powder). Paraformaldehyde can be solved in 1 X PBS for approx. 20-30 minutes @ 65 to 70 °C.
The sepharose matrix was purchased from the IBA company, Göttingen (http://www.iba-go.com/).
The refereence for the method: Herzberg, C., Flórez Weidinger, L. A., Dörrbecker, B., Hübner, S., Stülke, J. & Commichau, F. M. (2007) SPINE: A method for the rapid detection and analysis of protein-protein interactions in vivo. Proteomics 7: 4032-4035. PubMed
Other studies that made use of SPINE
- Commichau, F. M., Herzberg, C., Tripal, P., Valerius, O., and Stülke, J. (2007) A regulatory protein-protein interaction governs glutamate biosynthesis in Bacillus subtilis: The glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC. Mol Microbiol 65: 642-654. PubMed
