Difference between revisions of "SPINE"
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| − | ==[http://www.iba-lifesciences.com/ | + | ==[http://www.iba-lifesciences.com/IBA-Applications-Protein-interaction-SPINE-Technology.html '''See the principle''' ]== |
=='''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. | ||
The [[GltC]] protein was expressed carrying a Strep-tag and [[RocG]] expression was induced by arginine ([http://www.ncbi.nlm.nih.gov/sites/entrez/17608797 PubMed]). Expression of the Strep-tagged GltC protein allows to test the functionality of the protein. | The [[GltC]] protein was expressed carrying a Strep-tag and [[RocG]] expression was induced by arginine ([http://www.ncbi.nlm.nih.gov/sites/entrez/17608797 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. | 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- | + | 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 ([http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=+17994626 PubMed]). | 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. The interaction partner/s were identified by mass spectroscopy and Western blotting. | 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. | ||
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=='''SPINE for membrane proteins:'''== | =='''SPINE for membrane proteins:'''== | ||
| − | <pubmed> 21472855 </pubmed> | + | <pubmed> 21472855 24300168 </pubmed> |
| − | =='''Studies that made use of SPINE'''== | + | =='''Studies that made use of SPINE:'''== |
| − | + | <pubmed> 17608797 19193632 20572937 20933603 21622759 21803996 21992469 22001508 22517742 23192352 22512862 24325460 24375102 24666271 25711804 </pubmed> | |
| − | <pubmed> 17608797 19193632 20572937 20933603 | ||
| − | =='''The use of SPINE in other microbes'''== | + | =='''The use of SPINE in other microbes:'''== |
| + | <pubmed> 21123179 </pubmed> | ||
Latest revision as of 09:18, 9 March 2015
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:
Studies that made use of SPINE:
The use of SPINE in other microbes:
