The coding sequence of AIB1 was clonded into pCR3.1 at downstream of a T7 promoter. E. coli extract-based cell free in vitro expression of AIB1 protein was performed using the S30 T7 high yield Protein Expression System (Promega) following the manufacturer's protocol. For GST pull-down assays, 1 μg of E. coli-produced GST or GST fusion proteins were immobilized on glutathione-agarose (Roche) for 1 h at room temperature. After several washes, the agarose was resuspended and then incubated with in vitro expressed proteins or cell lysates containing indicated proteins at 4°C. After extensive washes, bound proteins were eluted, resolved with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by immumoblotting.
Glutathione agarose
Manufactured by Roche
Glutathione-agarose is a chromatographic resin used for the purification of proteins containing glutathione S-transferase (GST) tags. It consists of glutathione covalently linked to an agarose matrix. The glutathione moiety binds to the GST tag, allowing the target protein to be captured and purified from complex mixtures.
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Lab products found in correlation
2 protocols using glutathione agarose
1
In Vitro Expression and GST Pull-Down of AIB1
2
In Vitro Expression and GST Pull-Down of AIB1
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The coding sequence of AIB1 was clonded into pCR3.1 at downstream of a T7 promoter. E. coli extract-based cell free in vitro expression of AIB1 protein was performed using the S30 T7 high yield Protein Expression System (Promega) following the manufacturer's protocol. For GST pull-down assays, 1 μg of E. coli-produced GST or GST fusion proteins were immobilized on glutathione-agarose (Roche) for 1 h at room temperature. After several washes, the agarose was resuspended and then incubated with in vitro expressed proteins or cell lysates containing indicated proteins at 4°C. After extensive washes, bound proteins were eluted, resolved with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by immumoblotting.
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