and Vogelstein, 1983). At a specific activity of 3000 Ci/mmol, that 50uCi translates to 16.6 femtomoles of P dNTP being added to the reaction mix, while 50 uCi of aP labeled dNTP at a specific activity of 6000 Ci/mmol will add only 8.3 femtomoles to the reac tion. Unless sufficient unlabeled dntP is added the lower mass of the hotter dNTP solution added might end up slowing the random prime reaction down, giving the resulting probe a lower specific activity than the probe that used the 3000Ci/mmol Label Location on the Compound Consider the reason for using a radioactive molecule. Is the reaction involved in the transferring of the radioactive moiety to a biomolecule, such as a nucleic acid, peptide, or protein? Is the in vivo catabolism of the molecule being studied, perhaps in an ADME(absorption, distribution, metabolism, and excretion) study? Or perhaps the labeled molecule is simply being used as a tracer. For any situation, it,'s worthwhile to consider the following impacts of the label location: First, will the labels location allow the label or the labeled ligand to be incorporated? Next, once incorporated, will it produce the desired result or an unwanted effect? For example, will the label's presence in a nucleic acid probe interfere with the probe's ability to hybridize to its target DNA? The latter issue is also discussed in greater detail in Chapter 14, Nucleic Acid Hybridization. There are some reactions where the location of label is not criti- cal. a thymidine uptake assay is one such case. The labeling will york just as effectively whether the tritium is on the methyl group ng. The Form and Quantity of the radioligand The radionuclide is usually available in different solvents. The two main concerns are the effect (if any) of the solvent on the reaction or assay, and whether the radioactive material will be used quickly or over a long period. For example, a radiolabeled compound supplied in benzene or toluene cannot be added directly to cells or to an enzyme reaction without destroying the biological systems; it must be dried down and brought up in a com- patible solvent. Likewise a compound shipped in simple aqueous solvent might be added directly to the reaction, but might not be Becquerels, divide by 27(27.027): 50 uCi=50 x 10-Ci=(50 10°Ci×3.7×l00 dps/Ci)=1.85×10°dps=1.85×10°Bq= 185MBq.) 146and Vogelstein, 1983). At a specific activity of 3000 Ci/mmol, that 50mCi translates to 16.6 femtomoles of 32P dNTP being added to the reaction mix, while 50mCi of a 32P labeled dNTP at a specific activity of 6000Ci/mmol will add only 8.3 femtomoles to the reac￾tion. Unless sufficient unlabeled dNTP is added, the lower mass of the hotter dNTP solution added might end up slowing the random prime reaction down, giving the resulting probe a lower specific activity than the probe that used the 3000Ci/mmol material. Label Location on the Compound Consider the reason for using a radioactive molecule. Is the reaction involved in the transferring of the radioactive moiety to a biomolecule, such as a nucleic acid, peptide, or protein? Is the in vivo catabolism of the molecule being studied, perhaps in an ADME (absorption, distribution, metabolism, and excretion) study? Or perhaps the labeled molecule is simply being used as a tracer. For any situation, it’s worthwhile to consider the following impacts of the label location: First, will the label’s location allow the label or the labeled ligand to be incorporated? Next, once incorporated, will it produce the desired result or an unwanted effect? For example, will the label’s presence in a nucleic acid probe interfere with the probe’s ability to hybridize to its target DNA? The latter issue is also discussed in greater detail in Chapter 14, “Nucleic Acid Hybridization.” There are some reactions where the location of label is not criti￾cal. A thymidine uptake assay is one such case. The labeling will work just as effectively whether the tritium is on the methyl group or on the ring. The Form and Quantity of the Radioligand The radionuclide is usually available in different solvents. The two main concerns are the effect (if any) of the solvent on the reaction or assay, and whether the radioactive material will be used quickly or over a long period. For example, a radiolabeled compound supplied in benzene or toluene cannot be added directly to cells or to an enzyme reaction without destroying the biological systems; it must be dried down and brought up in a com￾patible solvent. Likewise a compound shipped in simple aqueous solvent might be added directly to the reaction, but might not be 146 Volny Jr. Becquerels, divide by 27 (27.027): 50mCi = 50 ¥ 10-6Ci = (50 ¥ 10-6Ci ¥ 3.7 ¥ 1010dps/Ci) = 1.85 ¥ 106dps = 1.85 ¥ 106Bq = 1.85MBq.)