The Yeast Two-Hybrid System:
an assay for protein-protein interaction
Outline of the two-hybrid system :
£»The Gal4 DNA binding domain is fused to MTR10.
£»The Gal4 Activation domain is fused to pGAD-hnRNPA1-£GM9
or pGAD-hnRNPA1-£GRG.
£»If MTR10 interacts with either pGAD-hnRNPA1-£GM9
or pGAD-hnRNPA1-£GRG, £]-galactosidase assay
will show positive results.
protocol
1.preparation of yeast competent cells
2.cotransformation (Y190)
| DNA binding domain | activation domain | |
| test | pDBMTR10(Trp-) | pGAD-A1£GM9 or £GRG(Leu-) |
| positive control | pDBMTR10(Trp-) | pGAD-A1FL(Leu-) |
| negative control | pDBMTR10(Trp-) | pGAD(Leu-) |
3.£]-galactosidase lift assay
