Preparation method of biotinylated protein and detection method using the same

A method for detecting a biomolecule interaction using Amplified Luminescence Proximity Homogeneous Assay, comprising the steps of:

• 1) preparing a protein containing a biotin tag in a cell-free protein synthetic method,
• 2) activating the protein containing the biotin tag with a biotinylating enzyme and a biotin derivative to prepare a biotinylated protein,
• 3) bonding directly or indirectly a biological molecule which possibly interacts with a protein to Acceptor beads, and
• 4) contacting the biotinylated protein prepared in the step 2), the Acceptor beads prepared in the step 3), and Donor beads to which streptavidin is bonded to cause a signal change due to the proximity of the Acceptor beads and the Donor beads, thereby to detect an interaction of the protein with the biological molecule.

A method for screening an autophosphorylated protein using Amplified Luminescence Proximity Homogeneous Assay, comprising the steps of:

• 1) preparing a candidate autophosphorylated protein containing a biotin tag in a cell-free protein synthetic method,
• 2) activating the candidate autophosphorylated protein containing the biotin tag with a biotinylating enzyme and a biotin derivative to prepare a biotinylated candidate autophosphorylated protein,
• 3) bonding directly or indirectly a phosphorylated detection antibody to Acceptor beads, and
• 4) contacting the biotinylated candidate autophosphorylated protein prepared in the step 2), the Acceptor beads prepared in the step 3), and Donor beads to which streptavidin is bonded to cause a signal change due to the proximity of the Acceptor beads and the Donor beads, thereby to detect a phosphorylation activity.