A simple protocol to detect interacting proteins by GST pull down assay coupled with MALDI or LC-MS/MS analysis.
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.
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Table 1 Trouble shooting
Posted 12 Oct, 2015
A simple protocol to detect interacting proteins by GST pull down assay coupled with MALDI or LC-MS/MS analysis.
Posted 12 Oct, 2015
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.
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