Radiolabel pulse chase

A useful adjunct is the well-known pulse-chase technique, in which exposure to a radiolabeled compound for a specified period of time (pulse) is followed by the administration of the same compound that is not labeled (chase). 

In a review of feeding and digestion in the Bivalvia [37], it was proposed that the accumulation of metal-bound particulates in the digestive gland was a two-phase process reflecting extracellular and intracellular digestion, and Viarengo [87] has reached similar conclusions. In a pulse chase study of the uptake of radiolabelled metals (Ag, Cd, Cr, Hg, Se) by the zebra mussel Dreissena... 

Pulse-chase experiments are particularly useful for tracing changes in the intracellular location of proteins or the transformation of a metabolite Into others over time. In this experimental protocol, a cell sample Is exposed to a radiolabeled compound—the pulse —for a brief period of time, then washed with buffer to remove the labeled pulse, and finally incubated with a nonlabeled form of the compound— the chase (Figure 3-36). Samples taken periodically are assayed to determine the location or chemical form of the radiolabel. A classic use of the pulse-chase technique was In studies to elucidate the pathway traversed by secreted proteins from their site of synthesis in the endoplasmic reticulum to the cell surface (Chapter 17). 

Several experiments have indicated that the ER is the site of addition of the bulk of TG to apo B. First, the size distribution of lipoproteins is the same in the lumen of the ER and Golgi of rat liver, and in newly secreted lipoproteins (J.E. Vance, 1993). Furthermore, a series of pulse-chase radiolabeling experiments in rat hepatoma cells indicate that the majority of TG associates with apo B in the ER (H.N. Ginsberg, 2003). On the other hand, immuno-electron microscopy studies in rat liver (R.J. Havel, 1976), and pulse-chase radiolabeling experiments in chicken hepatocytes (M.D. Lane, 1990) and rat hepatoma cells (E.A. Fisher, 2003), have suggested that the majority of TG assembles with apo B in the Golgi apparatus. The reasons for these conflicting conclusions remain unclear. 

To check if RNA turnover was influencing our assays, pulse-chase experiments were carried out (Table I, column II). When 5 min pulselabeling periods were followed by a 5 min chase, the amount of radiolabel in psbD-psbC transcripts declines significantly (13% to 53%). PsbD-psbC RNAs transcribed in plastids of 8 day-old dark-grown plants were less... 

N]-, [ C]-, pHjleucine or p Sjmethionine in the case of proteins) for various periods, after which the cells are lysed and the protein of interest is purified (often by immunoprecipitation with specific antibodies). The time course of isotope incorporation gives information about the rate (slope of curve) and extent (amplitude of curve) of the proteins synthesis. To measure degradation, cells are first pulse-labeled (i.e., exposed to radiolabeled precursor for a fixed period, after which sufficient nonla-beled precursor is added to reduce the radiospecific activity of the precursor). Then, the cells are further incubated, and the radiospecific activity of a particular protein of interest is determined (again usually after immunoprecipitation or some other means for achieving its isolation from other cellular proteins). The key point is that the chase allows one to stop radiolabel uptake almost instantaneously, thereby permitting the kinetic... 

Diagram showing the addition of radiotracer A at t = 0 (termed the "pulse") and the subsequent addition of excess unlabeled A (termed the "chase"). The relative size of the lettering serves to indicate the time-course of radiolabeled entry and exit from each metabolic pool. 

In some of the earliest studies of phospholipid transport between the inner and the outer membranes of E. coli, radiolabeling of PE revealed that the specific radioactivity of this lipid was 5-fold higher in the inner membrane than the outer membrane, immediately following a 30-s pulse with [ HJglycerol (A.M. Donohue-Rolfe, 1980). During the chase period, the specific radioactivity of the outer membrane increased, while that of the inner membrane decreased. After several minutes, the specific activities of both membranes asymptotically approached the same value, which indicated radioequilibration between the membranes. The ty2 for the translocation of PE was determined to be 2.8 min. The transport in these studies was independent of protein synthesis, lipid synthesis, and ATP synthesis. It appeared, however, to be dependent upon the cell s proton motive force. Analysis of LPS... 

Following UV irradiation (254 nm, 45 J/m2) of cultured hepatocytes [6] repair patches were radiolabeled with [methyl- HJthymidine for either 20 min (pulse period), 120 min (20 min pulse followed by a 100 min chase period with unlabeled thymidine), or 180 min (20 min pulse followed by 160 min chase). At the end of these treatments, repair incorporated radioactivity was quantified in MOPS-DNA and random DNA... 

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