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]]:''' == | ||
| − | 1 litre of a ''B. subtilis'' culture was grown to an | + | 1 litre of a ''B. subtilis'' culture was grown to an OD<sub>600</sub> 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 cells were harvested and washed once in 1 X PBS pH 6.5. | ||
The pellets can then be stored @ -20 °C. | The pellets can then be stored @ -20 °C. | ||
Revision as of 08:55, 9 January 2014
SPINE is a method to detect in vivo protein-protein interactions PubMed
Contents
- 1 See the principle
- 2 A detailed protocol to detect the interaction between RocG and GltC:
- 3 Relevant plasmids:
- 4 Biotin-containing proteins that are purified with the Strep-Tactin column
- 5 The reference for the method:
- 6 SPINE for membrane proteins:
- 7 Studies that made use of SPINE:
- 8 The use of SPINE in other microbes:
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; http://en.wikipedia.org/wiki/Paraformaldehyde). 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:
Studies that made use of SPINE:
The use of SPINE in other microbes:
Jens F Novak, Marit Stirnberg, Benjamin Roenneke, Kay Marin
A novel mechanism of osmosensing, a salt-dependent protein-nucleic acid interaction in the cyanobacterium Synechocystis Species PCC 6803.
J Biol Chem: 2011, 286(5);3235-41
[PubMed:21123179]
[WorldCat.org]
[DOI]
(I p)
