In-gel assay of ribonuclease A (A) alkaline phosphatase (B) and polynucleotide kinase (C) activities using an oligonucleotide substrate (5′ ACAGUAUUUG; Acr #1) chemically linked to the polyacrylamide matrix. The substrate used in A and B was radiolabeled at the 5′ end prior to incorporation into the gels, whereas in C the substrate was unlabeled. The diagrams that are part of each panel provide a schematic illustration of the corresponding reactions. BAP and PNK are abbreviations for bacterial alkaline phosphatase and polynucleotide kinase, respectively. The gels on the right of each panel have been silver stained and the sizes of marker bands (M) are indicated on the left of each gel. Autoradiographs on the left of each panel show the results of each in-gel assay. The amount of enzyme added to each lane is indicated at the top of the gels and autoradiographs. In D, the RNase A assay was repeated using fivefold more substrate in the absence of a reducing agent and followed the protocol of Bravo et al. (1994). The short and long incubation times were 30 and 90 min, respectively.
