phosphorus-radioisotopes has been researched along with cytidine-3--5--diphosphate* in 2 studies
2 other study(ies) available for phosphorus-radioisotopes and cytidine-3--5--diphosphate
| Article | Year |
|---|---|
3'-end labeling of RNA with [5'-32P]Cytidine 3',5'-bis(phosphate) and T4 RNA ligase 1.
This protocol is used to radiolabel the 3' ends of RNAs, either synthesized by in vitro transcription or purified from cells or tissues, by ligation of [5'-(32)P]cytidine 3',5'-bis(phosphate) (pCp). [5'-(32)P]pCp can be obtained commercially or prepared in the laboratory using polynucleotide kinase to phosphorylate cytidine-3'-monophosphate (Cp) with [γ-(32)P]ATP. "Homemade" [5'-(32)P]pCp is considerably cheaper and has a higher final concentration than that obtained from commercial sources. The labeling protocol uses T4 RNA ligase 1, which covalently joins [5'-(32)P]pCp to the free 3' hydroxyl of RNA. For best labeling, [5'-(32)P]pCp should be at least equimolar or higher to available 3'-hydroxyl ends. The reaction requires overnight incubation at low temperature. At the end of the procedure, the reaction is desalted by gel filtration to remove any unincorporated [5'-(32)P]pCp. Topics: Bacteriophage T4; Cytidine Diphosphate; Phosphorus Radioisotopes; RNA; RNA Ligase (ATP); Staining and Labeling; Viral Proteins | 2014 |
Regeneration of enzyme activity after western blot: activation of RNase L by 2-5A on filter--importance for its detection.
A rapid and convenient new procedure for detecting RNase L activity following Western blot by renaturation of the enzyme on the nitrocellulose sheets is described. This method allows the simultaneous analysis of enzymatic activity (e.g., cleavage of poly(uridylic acid)-3'-[32P]pCp) and RNase L binding to radioactivE probes (e.g., 2-5A-3'-[32P]pCp) in the same sample. Unlike previously published methods, this procedure eliminates interference from proteases or other RNases during the analysis of RNase L activity. The detection of RNase(s) L is also affected by the presence of endogenous 2-5A, 2-5A derivatives, or other possible "inhibitors" in cell extracts; this Western blot assay allows of RNase(s) L to be detected independently of intracellular 2-5A or analogs. Differences between the procedures used so far and this Western blot technique can indeed be demonstrated. It is shown with this Western blot assay that although RNase L has been described as a protein of 185-200 kDa under nondenaturating conditions, its 80-kDa (and 40-kDa) component is able to bind 2-5A and to cleave poly(uridylic acid) in a 2-5A-dependent way, independently of other subunit(s) or cofactor(s). Topics: Adenine Nucleotides; Animals; Blotting, Western; Cells, Cultured; Collodion; Cytidine Diphosphate; Endoribonucleases; Enzyme Activation; Humans; Male; Mice; Oligoribonucleotides; Phosphorus Radioisotopes; Poly U | 1991 |