Adventitious binding of 3H-labeled nucleotides to protein during polymerase assays.
The results of these studies demonstrate that 3H-labeled nucleotides and nucleosides are capable of binding to proteins such as bovine serum albumin or bulk proteins of cytoplasmic extracts in a time-, temperature- and concentration-dependent manner when incubated in phosphate buffer or under conditions for the assay of DNA polymerases. The binding of the labeled nucleotide to the protein is stable to washing with 10% cold trichloroacetic acid/1% sodium pyrophosphate and 95% ethanol. The radioactivity bound to the protein is rendered acid-soluble by treatment with pronase. Such non-specific binding can be a significant source of artifactual labeling in DNA or RNA polymerase assays.
D000818:Animals; D003600:Cytosol; D004257:DNA Polymerase II / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D013696:Temperature; D013942:Thymine Nucleotides / Q000378:metabolism*; D014316:Tritium; D014327:Trophoblasts / Q000201:enzymology
D003854:Deoxyribonucleotides; D011506:Proteins; D013942:Thymine Nucleotides; D014316:Tritium; D012710:Serum Albumin, Bovine; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate