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 '' | + | 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 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. | ||
| Line 13: | Line 13: | ||
Crude extracts (10-15 ml) were prepared by using a French Press. | 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 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 | + | 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 ([http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=+17994626 PubMed]). |
| − | A 12.5% SDS gel was loaded with the samples and the proteins were then visualized by silver-staining. | + | 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 1X PBS pH 6.5): | Preparation of the formaldehyde stock solution (max. 4% in 1X PBS pH 6.5): | ||
| − | We use | + | 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/). | The sepharose matrix was purchased from the IBA company, Göttingen (http://www.iba-go.com/). | ||
Revision as of 12:15, 25 February 2010
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 1X 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: pGP380, pGP382
for use in E. coli: pGP172, pGP574
Biotin-containing proteins that are purified with the Strep-Tactin column
The reference for the method:
Other studies that made use of SPINE
