Pulse-chase protein production

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To determine the amount of protein generated during a given period of time, whether bulk protein in the cell or a particular protein from a gel or other separation technique, grow the cell with radiolabelled amino acids for that time, then resuspend the cells in medium lacking the radioactive amino acids.  At time points afterwards, beginning with immediately after resuspension, isolate protein from the cells and measure its concentration (say by a Bradford assay) and the radioactivity of your preparation.  The radioactivity per unit of protein immediately after the end of labelling tells you how strongly the protein was labelled during that period.  The decrease in radioactivity per unit of protein over time tells you how fast the protein is turned over.
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It may be important to take the supernatant of the culture and measure the radioactivity of its protein fraction to know about secreted protein.
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[[Category:Protocol]] [[Category:Needs attention]]

Current revision

To determine the amount of protein generated during a given period of time, whether bulk protein in the cell or a particular protein from a gel or other separation technique, grow the cell with radiolabelled amino acids for that time, then resuspend the cells in medium lacking the radioactive amino acids. At time points afterwards, beginning with immediately after resuspension, isolate protein from the cells and measure its concentration (say by a Bradford assay) and the radioactivity of your preparation. The radioactivity per unit of protein immediately after the end of labelling tells you how strongly the protein was labelled during that period. The decrease in radioactivity per unit of protein over time tells you how fast the protein is turned over.

It may be important to take the supernatant of the culture and measure the radioactivity of its protein fraction to know about secreted protein.

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