| Popis: |
Publisher Summary This chapter presents a protein kinase assay (enzyme activity dot blot) in which both the kinase and its substrate are immobilized on nitrocellulose. This assay, originally worked out for casein kinase II, is comparable to the standard liquid assay with respect to sensitivity and linearity and has a number of advantageous features. The samples containing the protein kinase activity to be assayed are immobilized on a nitrocellulose filter, either by manual spotting or by vacuum filtration using a commercial slot or dot blotter. The filter is then incubated in an appropriate protein substrate, which binds to remaining binding sites on the filter, and then with radioactive adenosine triphosphate (ATP). The reaction is stopped and unutilized ATP simultaneously removed simply by washing the filter. Incorporation is determined by autoradiography and/or liquid scintillation counting. Most problems with the technique can be traced to overloading the nitrocellulose paper, either with total protein or with enzymatic activity. Care must be taken to avoid excessive input of protein kinase activity because the dot assay begins to deviate from linearity beyond about 50 p M units/dot. Additionally, bleeding of high-activity dots may contaminate adjacent dots on the nitrocellulose. |
