Difference between revisions of "SPINE"

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(A detailed protocol to detect the interaction between RocG and GltC:)
(Studies that made use of SPINE:)
 
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==[http://www.iba-lifesciences.com/protein_interaction_spine_Technology.html '''See the principle''' ]==
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==[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 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.  
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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-lifesciences.com/protein_interaction_spine_Technology.html).
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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>
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<pubmed> 21472855 24300168 </pubmed>
  
=='''Other studies that made use of SPINE'''==
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=='''Studies that made use of SPINE:'''==
'''Additional references:''' {{PubMed|22512862}}
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<pubmed> 17608797  19193632 20572937 20933603 21622759 21803996 21992469 22001508 22517742 23192352 22512862 24325460 24375102 24666271 25711804 </pubmed>
<pubmed> 17608797  19193632 20572937 20933603 21123179 21622759 21803996 21992469 22001508 23192352 </pubmed>
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=='''The use of SPINE in other microbes:'''==
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<pubmed> 21123179 </pubmed>

Latest revision as of 09:18, 9 March 2015

SPINE is a method to detect in vivo protein-protein interactions PubMed


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

PycA, AccB

The reference for the method:


SPINE for membrane proteins:


Studies that made use of SPINE:


The use of SPINE in other microbes: