This is a protocol preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Method Article
Asis Datta
Asis Datta
Corresponding Author
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.
Keywords
interacting proteins,Glutathione-S-transferase ,MALDI,LC-MS/MS ,in gel trypsin digestion,pull-down assay
The authors declare no competing financial interests.
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.doc
Table 1 Trouble shooting
Loading...
Version History
CURRENT STATUS: Posted
Version 1
Posted 12 Oct, 2015
No community comments so far
Metrics
Comments: 0
HTML Views: ...
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)
Method Article
Asis Datta
Asis Datta
Corresponding Author
Version 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.
Loading...
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)