Sigma′s Plant Fractionated Protein Extraction Kit is designed specifically for use in plant bioscience to extract qualitative hydrophilic and hydrophobic proteins samples from any type of plant species or tissue for downstream proteomic applications. The protocol does not require any ultracentrifugation or aqueous polymer two-phase partitioning (APTP). The kit includes five reagents; a plant specific protease inhibitor, a specially formulated reagent to extract hydrophilic proteins and a new chaotropic reagent with increased solubilizing power to extract more hydrophobic proteins. Also included in the kit are reducing reagent Tributylphosphine (TBP) and alkylating reagent, Iodoacetamide. These reagents improve separation during isoelectric focusing and 2-D gel electrophoresis. The protease inhibitor is a mixture of protease inhibitors with broad specificity for the inhibition of serine, cysteine, aspartic, metalloproteases and aminopeptidases. The cocktail contains 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), bestatin, pepstatinA, E-64, leupeptin, and 1,10-phenanthroline.
Following removal of polyphenolics, tannins and other interfering substances, ground plant tissue, fresh or frozen, is resuspended in a specially formulated reagent to extract hydrophilic proteins. After sequential extractions of hydrophilic proteins, addition of the chaotropic reagent provided extracts hydrophobic membrane bound proteins. Plant debris is pelleted by centrifugation and protein extract solutions are collected. The end results are qualitative fractionated protein samples, ready for downstream proteomic analysis.