View source for SPINE

'''SPINE is a method to detect ''in vivo'' protein-protein interactions''' [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=+17994626 PubMed]


==[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]]:''' ==

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 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. 
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 ([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. 

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:'''==
<pubmed> 17994626 </pubmed>

=='''SPINE for membrane proteins:'''==
<pubmed> 21472855 24300168 </pubmed>

=='''Studies that made use of SPINE:'''==
<pubmed> 17608797  19193632 20572937 20933603 21622759 21803996 21992469 22001508 22517742 23192352 22512862 24325460 24375102 24666271 25711804 </pubmed>

=='''The use of SPINE in other microbes:'''==
<pubmed> 21123179 </pubmed>