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Sample Preparation Solutions for Cellular Protein Fractionation Poster, Rev A


Mouse liver tissue samples were processed with three different sample preparation kits to extract and enrich specific subclasses of proteins. Instructions for each kit were followed exactly. Protease inhibitors were added to the starting buffers immediately prior to use to prevent proteolysis during extraction. Following extraction, if required, protein samples were processed with the ReadyPrep 2-D cleanup kit to remove salts and detergents and to create a low-conductivity sample suitable for isoelectric focusing (IEF). This cleanup kit was also employed, if required, to concentrate proteins from dilute samples. Prior to IEF all samples were solubilized in PSB, to which 50 mM DTT, 2 mM TBP, and 0.2% Bio-Lyte ampholytes (310) were added, and applied to 17 cm, pH 310 NL, ReadyStrip IPG strips. Approximately 455 g of protein was loaded to each strip. E. coli samples were processed with the ReadyPrep membrane II kit protocol. Extracted membrane proteins were applied to 24 cm, pH 47 ReadyStrip IPG strips.


2-D Electrophoresis
The IPG strips were focused using a PROTEAN IEF cell for ~60,000 V-hr at a final focusing voltage of 10,000 V. The cell was set for rapid voltage ramping. Focused IPG strips were loaded onto 816% SDS-PAGE gels following equilibration for 10 min with DTT containing buffer followed by 10 min with iodoacetamide containing buffer. Following electrophoresis, gels were fixed for 30 min, stained with Bio-Safe Coomassie stain for 1 hr, and then destained for at least 2 hr before scanning with a GS-800 densitometer.


Protein Identification
2-D gel images were processed for analysis with PDQuest 2-D analysis soft
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