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Fluorescence Microplate-Based Cycloheximide Chase Assay: A Technique to Monitor the Degradation Kinetics of Fluorescent Nuclear Misfolded Proteins
Transkript
After seeding and transfecting HeLa cells in 96-well plates according to the text protocol; 20 to 24 hours after transfection, examine the cells for GFP expression. Remove the medium at approximately 200 microliters of 1x PBS to each well, and then, aspirate it to remove the residual DMEM.
Add 60 microliters of low-fluorescence DMEM with 5% FBS, and 50 micrograms per milliliter of cycloheximide, and add 10 micromolar MG132 to one set of samples.
With a fluorescence plate reader, measure the GFP signal reading the plates every hour for up to 8 to 10 hours. Export the data, and carry out statistical analysis according to the text protocol.
