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Section 3.4.6


Describe in detail experiments or other methods of collecting data. The purpose of the procedure section is to allow a reader of the report to reproduce the experiment or data collection process.

The procedure section should be written in narrative form with illustrations of all test setups and procedures included within the text (see Integrating Graphics and Text).

As in all narratives, organize the material to follow the actual sequence of events. Separate each group of actions into one or more paragraphs, and describe each discrete action in one or more sentences.

List all materials and apparatus used in the procedures in sufficient detail that a reader could reproduce the experiment.

Experimental Procedures

CNBr mapping was performed on immunoprecipitated pleckstrin which was gel-purified by SDS-polyacrylamide gel electrophoresis. A gel slice containing the phosphopleckstrin was mixed with 50 mM ammonium carbonate (pH 8.5), 0.1% SDS, 1% mercaptoethanol. Pleckstrin contained in the eluate was passed through glass wool, trichloroacetic acid-precipitated, washed twice with a 50:50 mixture of cold ethanol:ether, and then vacuum-dried. The sample was resuspended in 30 µl of 50 mg/ml CNBr in 70% formic acid and incubated at room temperature for 1 h. At this point, the digest was lyophilized with 1 ml of distilled water and again vacuum-dried. When noted, the CNBr fragments were further digested by incubating them overnight in the dark at room temperature in 30 µl of 10 mg/ml iodosobenzoate (IBZO) in 80% acetic acid with 4 M guanidine HCl(26). All samples were then lyophilized with 1 ml of distilled water, vacuum-dried an additional three times, and fractionated on a Tricine gel(27).

--S. Abrams et al., "Protein Kinase C Regulates Pleckstrin by Phosphorylation of Sites Adjacent to the N-terminal Pleckstrin Homology Domain," Journal of Biological Chemistry

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