Phosphorus autoradiography

Also present in the first test tube is a synthetic analog of ATP in which both the 2 and 3 hydroxyl groups have been replaced by hydrogens This compound is called 2 3 dideoxyadenosme triphosphate (ddATP) Similarly ddTTP is added to the second tube ddGTP to the third and ddCTP to the fourth Each tube also contains a primer The primer is a short section of the complementary DNA strand which has been labeled with a radioactive isotope of phosphorus ( P) When the electrophoresis gel is examined at the end of the experiment the positions of the DNAs formed by chain extension of the primer are located by a technique called autoradiography which detects the particles emitted by the P isotope... 

The examples for radioactive labeling by phosphorus-32 ( P) and iodine-125 ( 1) in this chapter were chosen for two reasons on one hand, they are relatively easy to do, and on the other hand, the measurement of radioactivity is simple. is counted in water in a liquid scintillation counter by measuring the Cerenkov radiation and is measured in a gamma counter. Both isotopes may be detected also by autoradiography. A further advantage of both isotopes is their short half-life, which eases the disposal of nuclear waste. 

Subsequent to recovery of the total lipids of a cellular preparation as a chloroform-soluble fraction, the total phosphorus content can be determined (see Chapter 3) and then, depending on the amount of lipid phosphorus (or whether the preparation is radiolabeled or not, see below), analytical and/or preparative thin-layer chromatography can be undertaken. In either case, if the experimental protocol is centered on a signal-transduction process, then there may be insufficient material for a phosphorus analysis. In the latter instance, the cellular preparation is prelabeled with 32P or [3H]inositol and the labeled products are located by autoradiography. A preferred type of adsorbent (for thin-layer chromatography) is Merck silica gel 60 (oxalate impregnated). An effective solvent for separation of the phosphatidylinosi-tols and other lipids is chloroform-acetone-methanol-acetic acid-water (80 30 26 24 14, v/v). The approximate / values of cellular phospholipids under these conditions are presented as follows ... 

Fig. 3.1. Simultaneous bidirectional transport of phosphorus within mycelium of Hypholoma fasciculare demonstrated using doublelabelling and digital autoradiography, (a) Experimental design, (b) Imaging of isotope distribution 29 days after addition of labels. Microcosms are 20 cm long. Derived from Lindahl et al. (2001).

In other studies, Edwards (248), Fairchild, Tonna, and Seibold (257, 258) and Frigero (293) have employed activation analysis low-background radiographic techniques to measure the boron distribution within cells in order to use Boron-10 in clinical trials of an experimental therapuetic procedure, that is, neutron capture therapy. These particular cellular distribution studies were carried out on mouse spleen, and, after the activation, the materials were exposed to photographic nuclear track emulsions of Eastman Kodak Type NTA. The countable a-particle tracks obtained in these experiments showed that concentrations of B-10 of 60 Mg/gm, the amount that can be safely delivered to tumors in human subjects, could be readily determined. In other applications of activation analysis and autoradiography, Vincent (963) and Vincent, Haumont and Roels (964) made microscopic determinations of sodium in compact bone and of bone phosphorus. 

In the chain termination method, a single-stranded DNA of unknown sequence is mixed with primer and divided into four separate reaction mixtures. To each reaction mixture are added all four deoxynucleoside triphosphates (dNTPs), one of which is labeled in the 5 phosphoryl group with phosphorus-32 ( P) so that the newly synthesized fragments can be visualized by autoradiography. 

Fuhs, G. W., and E. Canelli. 1970. Phosphorus-33 autoradiography used to measure phosphate uptake by individual algae. Limnol. Oceanogr. 15(6) 962-967. 

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