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Title 

A screening method of SH2 domain ligands and blockers using a solid phase binding

Authors 

Woo Suk KohSun Young YoonEun Kyung LeeByoung-Mog KwonJae Won KimMi Young Han

Publisher 

Elsevier

Issue Date 

1997

Citation 

Cancer Letters, vol. 120, no. 1, pp. 1-7

Keywords 

PhosphopeptideScreeningSH2glutathione transferaseligandamino acid sequencebinding competitionisotope labelingpriority journalprotein binding

Abstract 

We have developed a high throughput screening method for SH2 domain binding ligands and blockers. This method measures directly the binding of a 3H-labeled phosphopeptide derived from the sequence around tyrosine317 in the human Shc (SpYVNVK) to the SH2 domain of Grb2, which is precoated as glutathione S-transferase fusion proteins on solid phase. The optimum concentration for the fusion protein coating was 300 ng/100 μl/well for SH2 domain binding. Although an 8-h incubation at 4°C for the coating of fusion protein was required to reach a maximum binding, even a 2-h coating produced 84% of the maximum binding. Saturation of ligand peptide binding in our assay system was observed at 10 pmol/well for the SH2 domain. However, 2 pmol/well showed consistent and reproducible results for the binding when the incubations were performed for 8 h at 4°C. Competitive binding inhibition studies with various unlabeled phosphopeptides imply that the binding assay is highly specific to peptide sequences and able to screen possible ligands or blockers of signal transduction pathway mediated by Grb2 SH2 binding. In conclusion, our new method for SH2 domain binding is easy, rapid, and most of all inexpensive. These advantages over existing assay methods make this method especially suitable for a high throughput application, such as the screening for anticancer drug candidates.

ISSN 

0304-3835

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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