Optimizing a method of protein extraction for two-dimensional electrophoretic separation of proteins from planarians (Platyhelminthes, Turbellaria).
Different procedures for microscale extraction of proteins from small amounts of tissue of planarians (Platyhelminthes, Turbellaria) to be analyzed by two-dimensional gel electrophoresis (2-D PAGE) are compared. Three extraction methods were assessed: (i) extraction of soluble proteins with nondenaturing Tris buffers, (ii) extraction with Tris buffer containing the anionic detergent sodium dodecyl sulfate (SDS), and (iii) denaturing extraction under reducing conditions in the presence of urea and Nonidet P-40 (NP-40) with or without SDS. Buffers combining minute concentrations of SDS (0.01%), denaturing concentrations of urea (8M) and alkaline pH solubilized the greatest number of proteins without detectable proteolysis. Neither the presence of protease inhibitors nor higher concentrations of SDS improved protein extraction. We have applied this method to planarians to detect proteins specific to the pharynx. The resulting two-dimensional pattern shows a larger number of specific spots than in previous extraction methods.
Collet
J
J
Department of Genetics, Faculty of Biology, University of Barcelona.
Baguñá
J
J
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D008846:Micromanipulation; D010447:Peptide Hydrolases; D010614:Pharynx / Q000737:chemistry; D010932:Planarians / Q000737:chemistry*; D011480:Protease Inhibitors; D011489:Protein Denaturation; D011506:Proteins / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D013048:Specimen Handling / Q000379:methods*; D014508:Urea
D011480:Protease Inhibitors; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D010447:Peptide Hydrolases