SPINE
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:
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)
Studies that made use of SPINE:
Additional references: PubMed
The use of SPINE in other microbes:
