Analysis of intracellular modifications

A method for performing a Western blot for determining modification of an intracellular target protein in a transfected cell using a fusion protein serving as a surrogate protein for said-target protein, said surrogate protein comprising an enzyme donor (ED), wherein said ED is a member of an enzyme fragment complementation pair of β-galactosidase that forms an active enzyme with the complementary enzyme acceptor fragment (EA) and said surrogate protein is modified in said cell in the same manner as said target protein, said modification occurring as a result of changes in the environment of said cell where said modifications result in a change in the migration rate of said surrogate protein in gel electrophoresis, wherein said method comprises:

• lysing said transfected cell, transformed with an expression construct of said fusion protein resulting in intracellular expression of said fusion protein, to form a lysate;
• separating said lysate by gel electrophoresis into bands where said fusion protein and intracellular modifications thereof comprising said ED are separated by at least one of size and mass-to-charge ratio;
• transferring said bands to a membrane;
• adding EA and substrate to said membrane to visualize modified and unmodified fusion protein bands free of other bands;
• and
• determining from said bands whether said target protein is modified as a result of said changes in the environment of said cell.