Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

The overall goal of this procedure is to measure proteasome activity in living cells using A GFP Deron Fusion Protein first clone the custom oligonucleotides for Deron and Deron frameshift. Enter the P-E-G-F-P-C one vector and continue with the protocols for the gateway system in Vitrogen, then produce viruses for each construct using HEK 2 93 FT cells. After determining the titer transduced cells with the virus and apply antibiotic selection, then expand the resulting stable GFP DRON or GFP Dron frameshift expressing cells.

This technique facilitates experiments that monitor cell responses to given treatments through fluorescence signal measurements. For instance, Proteasome activity can be easily assessed using epi fluorescence or flow cytometry. The main advantage of this technique over existing methods like lumino or fluoro genic assays using whole cell is acids, is that the proteasome activity is measured in living cells For the transfection dilute 36 microliters of lipo 2000 reagent in 1.5 Milliliters of DMEM in another 15 milliliter falcon tube dilute the DNA constructs in 1.5 milliliters of DMEM.