Difference between revisions of "SPINE"

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(A detailed protocol to detect the interaction between RocG and GltC:)
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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 ''Bacillus subtilis'' culture was grown to an OD600 of approx. 1.0 and incubated with 0.6% formaldehyde (stock solution 4% in PBS, pH 6.5!) for 20 minutes @ 37°C on a shaker.  
+
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.
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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 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. We identified the interaction partner/s 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.  
  
 
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 paraformaldehyde (a white powder). Paraformaldehyde can be solved in 1 X PBS for approx. 20-30 minutes @ 65 to 70 °C.  
+
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


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

PycA, AccB

The reference for the method:


Other studies that made use of SPINE

Fabian M Commichau, Fabian M Rothe, Christina Herzberg, Eva Wagner, Daniel Hellwig, Martin Lehnik-Habrink, Elke Hammer, Uwe Völker, Jörg Stülke
Novel activities of glycolytic enzymes in Bacillus subtilis: interactions with essential proteins involved in mRNA processing.
Mol Cell Proteomics: 2009, 8(6);1350-60
[PubMed:19193632] [WorldCat.org] [DOI] (I p)

Fabian M Commichau, Christina Herzberg, Philipp Tripal, Oliver Valerius, Jörg Stülke
A regulatory protein-protein interaction governs glutamate biosynthesis in Bacillus subtilis: the glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC.
Mol Microbiol: 2007, 65(3);642-54
[PubMed:17608797] [WorldCat.org] [DOI] (P p)