General Description
The Plant Hydrophobic Protein Extraction Kit is designed specifically for use in plant bioscience to extract a qualitative sample of hydrophobic proteins from any type of plant species or tissue. The protocol does not require any ultracentrifugation or aqueous polymer two-phase partitioning (APTP). The kit includes three reagents, a plant specific protease inhibitor cocktail, a specially formulated reagent to extract hydrophilic proteins (Plant Protein Extraction Reagent Type 1), and a new chaotropic reagent with increased solubilizing power to extract more hydrophobic proteins (C0356, Protein Extraction Reagent Type 4). The protease inhibitor cocktail is a mixture of protease inhibitors with broad specificity for the inhibition of serine, cysteine, aspartic, metalloproteases, and aminopeptidases. It contains pepstatin A, E-64, leupeptin, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), bestatin, and 1,10-phenanthroline. The cocktail has been demonstrated to be highly effective in preventing protein degradation during the extraction process.
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 the hydrophilic proteins, the chaotropic reagent is used to extract hydrophobic membrane bound proteins. Plant debris is pelleted by centrifugation and the hydrophobic protein extract is collected. The end result is a qualitative hydrophobic protein sample, ready for downstream proteomic analysis.
