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Method Article
A simple protocol to detect interacting proteins by GST pull down assay coupled with MALDI or LC-MS/MS analysis.
Asis Datta
Asis Datta
Corresponding Author
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Glutathione-S-transferase (GST) tagged proteins have variety of applications including purification of fusion proteins. The GST pull-down assay is an in vitro method used to determine a physical interaction between a GST tagged probe protein to an unknown or a known target protein. The procedure involves incubation of the GST fusion protein immobilized on glutathione-agarose beads with the total cell lysate. Complexes eluted from the beads are resolved by SDS-PAGE and analyzed by silver/comassie staining. The protein bands are subjected to in gel trypsin digestion followed by MALDI (Matrix-assisted laser desorption/ionization) or LC-MS/MS (Liquid chromatography- mass spectrometry) analysis of resulting peptides to identify the interacting proteins.
Keywords
interacting proteins,Glutathione-S-transferase ,MALDI,LC-MS/MS ,in gel trypsin digestion,pull-down assay
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Associated Publications
A calmodulin like EF hand protein positively regulates oxalate decarboxylase expression by interacting with E-box elements of the promoter
by Ayushi Kamthan, Mohan Kamthan, Avinash Kumar, +5
Scientific Reports (12 October, 2015)
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This is a preprint. It has not completed peer review.
Method Article
A simple protocol to detect interacting proteins by GST pull down assay coupled with MALDI or LC-MS/MS analysis.
Asis Datta
Asis Datta
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 12 Oct, 2015
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Glutathione-S-transferase (GST) tagged proteins have variety of applications including purification of fusion proteins. The GST pull-down assay is an in vitro method used to determine a physical interaction between a GST tagged probe protein to an unknown or a known target protein. The procedure involves incubation of the GST fusion protein immobilized on glutathione-agarose beads with the total cell lysate. Complexes eluted from the beads are resolved by SDS-PAGE and analyzed by silver/comassie staining. The protein bands are subjected to in gel trypsin digestion followed by MALDI (Matrix-assisted laser desorption/ionization) or LC-MS/MS (Liquid chromatography- mass spectrometry) analysis of resulting peptides to identify the interacting proteins.
1 comment flagged for moderation
If you posted a comment and think it was mistakenly removed, please contact customer support at [email protected].
Associated Publications
A calmodulin like EF hand protein positively regulates oxalate decarboxylase expression by interacting with E-box elements of the promoter
by Ayushi Kamthan, Mohan Kamthan, Avinash Kumar, +5
Scientific Reports (12 October, 2015)