Figure 3. Y2H and ELISA based assays for interaction between MP and VPg.

(A) Y2H interaction between MP and VPg. pGBK T7 (MP and p53 ) and pGAD T7 (VPg and T Ag) clones were transformed in pairs into AH109 strain and plated on to –Leu-Trp SD transformation selection plates and incubated for 96 hrs. Colonies which grew were marked and again replica plated onto various nutritional marker SD plates having different stringency of reporter gene expression as shown in the figure. To determine α- galactosidase activity, colonies were plated onto SD plates having α-X Gal (last two columns). AH109 cells transformed with pairs of pGBK T7-MP and pGAD T7 or pGAD T7-VPg and pGBK T7 clones were also plated on to selection plates to rule out the possibility of auto activation of reporter genes (last two rows). (B) ELISA based interaction study between GST-MP and VPg. VPg (5 µg) (P1) coated ELISA plates were blocked with 10% skimmed milk in 1× PBS (Block) followed by the addition of 5 µg of GST-MP (P2), ELISA was performed with anti MP polyclonal antibody (pAb to P2) and developed using anti rabbit IGg and DMB H₂O₂ (sAb+Sub). Details of the steps and controls are marked in the figure; BSA and GST were used as the negative controls. The bar represents the absorbance of the samples measured at 450 nm.