Quantitation of Y2H interaction by ß-galactosidase assay and estimation of level of protein expression.
(A) The transformed colonies which showed positive Y2H interaction in the –Leu- Trp- His SD plates were grown in liquid cultures (-Leu –Trp –His medium) and were assayed for β-galactosidase activity to validate and quantify the two-hybrid interactions. For testing the interactions in the pairs that did not grow in -Leu –Trp –His SD plate, the cells grown on -Leu –Trp medium were used. The assay was performed as described in the methods section. The results are presented as the percentage in arbitrary units of ß-galactosidase activity (the values are indicated on the top of each bar) obtained for the interaction between p53 and TAg (100%). The values represent the mean of at least three separate experiments. (B) ELISA to check the expression of the proteins was performed by coating the total protein isolated from AH109 cells transformed with bait and pray plasmid and using c-Myc monoclonal or HA polyclonal antibody as the primary antibody. The bar represents the absorbance measured at 450 nm for each of the protein pairs. The level of expression of all the HA tagged SeMV proteins was comparable to the expression of cMyc tagged MP.