Assay Method Information |
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| Ribonuclease Activity Assay |
Description: | The Ribonuclease Activity assay used was adapted from the procedure described by Anfinsen et al. and Slifman et al. Determination of ribonuclease activity of RNase A and EDN proceeded as follows: yeast tRNA (Sigma-Aldrich) was solubilized in 50 mM sodium phosphate, pH = 7.4, at a concentration of 60 uM. Various tRNA concentrations were used: 2 uM, 3 uM, 4 uM, 5 uM and 6 uM. The final volume of the reaction was 300 ul. tRNA solutions were incubated at 30 C for 10 min. 10 nM of enzyme final concentration from a 0.6 uM stock solution added to the reactionmixture. The reaction was stopped one minute and five minutes later in the case of RNase A and EDN respectively, by the addition of 300 ul of fresh ice-cold solution of 40 mM lanthanum nitrate, 6% v/v perchloric acid. Stopped reactions were maintained on ice for 15 min, and insoluble tRNA was removed by centrifugation at 4 C for 15 min at 14.500 rpm on a bench centrifuge. The amountof cleaved tRNA was determined from the ultraviolet absorbance at 260 nm (A260) of the supernatant fraction. |
Affinity data for this assay | |
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