Difference between revisions of "Pulse-chase protein production"

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I don't know what this heading is supposed to mean. Labeling? 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.
  
When hearing this title, the first thing that would come to my mind is to use a synthetic gene regulatory network, perhaps a puslation or oscillator device, to control expression of a heterologous protein. Maybe such a system can be developed to help overcome inclusion body formation; a system which checks the level of chaperons before producing more recombinant protein. Thats my wild guess of what Pulse-chase could mean -Devin
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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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I am fairly certain that pulse-chase protein production refers to experiments known as pulse-labeling. This is when you subject your organism to an environmental stimulus (hot, cold, IPTG, etc.), you wait some amount of time, then introduce a radio-labeled amino acid (typically methionine). So the pulse is the stimulus and the chase is the label such as hot methionine. - Lon
 
  
 
[[Category:Protocol]] [[Category:Needs attention]]
 
[[Category:Protocol]] [[Category:Needs attention]]

Latest revision as of 03:32, 25 September 2007

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.