Glutathione S-transferase pull-down assays using dehydrated immobilized glutathione resin.
We have developed an affinity-precipitation technique to facilitate conducting glutathione S-transferase (GST) pull-down assays. The dehydrated immobilized glutathione resin format, when combined with microcentrifuge spin columns, is a powerful tool that enables the simultaneous performance of resin hydration, the binding of the GST fusion protein, and the pull-down step with the appropriate protein partner in a semihigh-throughput fashion (multiple samples processed at the same time). The entire assay process is shortened and recovery is enhanced when coupled with a spin-column format, providing a convenient way to study protein-protein interactions. We successfully tested the resin format/technique in three common pull-down applications utilizing radiolabeled, overexpressed, and activated endogenous interacting protein partners.
Ren
Ling
L
Bioresearch Division, Perbio Science, 2202 N Bartlett Avenue, Milwaukee, WI 53202-1009, USA.
Chang
Edith
E
Makky
Khadijah
K
Haas
Arthur L
AL
Kaboord
Barbara
B
Walid Qoronfleh
M
M
D001671:Biochemistry / Q000379:methods*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002498:Centrifugation / Q000295:instrumentation / Q000379:methods; D004268:DNA-Binding Proteins; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D025941:Protein Interaction Mapping / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D012117:Resins, Synthetic / Q000737:chemistry*; D013462:Sulfur Radioisotopes; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C121375:Pcbp1 protein, mouse; D011518:Proto-Oncogene Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012117:Resins, Synthetic; D013462:Sulfur Radioisotopes; D016159:Tumor Suppressor Protein p53; D044763:Ubiquitin-Conjugating Enzymes; D051736:Proto-Oncogene Proteins c-mdm2; D005982:Glutathione Transferase; D019908:Proto-Oncogene Proteins c-raf; D005978:Glutathione