Section 3.4.6

Procedure

Describe in detail experiments or other methods of collecting data. The purpose of the proceduresection 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 eachgroup 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 sufficientdetail that a reader could reproduce the experiment.

CNBr mapping was performed on immunoprecipitated pleckstrin which was gel-purified bySDS-polyacrylamide gel electrophoresis. A gel slice containing the phosphopleckstrin was mixed with50 mM ammonium carbonate (pH 8.5), 0.1% SDS, 1% mercaptoethanol. Pleckstrin contained inthe eluate was passed through glass wool, trichloroacetic acid-precipitated, washed twice with a 50:50mixture of cold ethanol:ether, and then vacuum-dried. The sample was resuspended in 30 µlof 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, theCNBr fragments were further digested by incubating them overnight in the dark at room temperaturein 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 threetimes, and fractionated on a Tricine gel(27).

--S. Abrams et al., "Protein Kinase C Regulates Pleckstrin by Phosphorylation of SitesAdjacent to the N-terminal Pleckstrin Homology Domain," Journal of BiologicalChemistry

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