Yeast two-hybrid assay of the interaction of different regions of SMC3 and SMC1 with Hinderin. A) SMC3 and Hinderin constructs in pGBKT7 are schematized on the left-hand side. All numerals refer to the amino acid sequence. The SMC3-465/807 construct harbors the protein hinge domain and was utilized as bait for the screening of the Clontech Matchmaker human cDNA library. SMC1, SMC3 and Hinderin constructs cloned in pACT2 or pGADT7 are illustrated on the right-hand side of the panel. H-47/577 represents the pACT2 clone retrieved from the screening. We scored the strength of interaction based on the rate of appearance of Blue colonies and the intensity of the color developed. The null score (-) was assigned when no blue colonies were visible after ten days. To obtain a quantitative measure of the rate of protein interaction, yeast colonies were grown in selective media and after lysis the β-galactosidase activity released in the supernatant assayed using a chromogenic substrate as detailed in the Methods. The results shown are the mean ± SD of three independent determinations.
Patel and Ghiselli BMC Cell Biology 2005 6:3 doi:10.1186/1471-2121-6-3