Prior studies have shown that 1 gram of dry tissue of G. max roots contains 2.5 mg of protein and 25 mg of starch [67,68]. Based on these published measurements, to remove starch, weighed root samples have been treated with porcine pancreas α-amylase (Sigma-Aldrich®). The procedure has required the addition of 25 units of amylase (capable of releasing 25 mg of starch) per 1 gram of sample. Amylase has been diluted in sufficient volume of 100 mM Tris-HCl buffer (pH 7.0) to completely react with the starch present within the studied samples amount. The de-starching reaction has been performed at 37°C for 3 hours. Subsequently, the solution has been centrifuged at 10,000 rpm for 5 minutes and the supernatant discarded. To each gram of de-starched root samples, 0.025 mg of Pierce Protease (Thermo Fisher Scientific ), capable of removing 2.5 mg of protein, along with sufficient amount of 50 mM acetic acid (pH 8.0) has been added to enzymatically react with the sample material with the reaction being carried out at 37°C for 4 hours. The solution has then been centrifuged at 4,000 rpm for 8 minutes, and the pellet has been washed with deionized water (10 ml). Upon removal of the supernatant, the pellet has been de-pectinated three times by boiling at 100° C in 50 mM EDTA (pH 6.8) for 15 minutes. The extracted pectin has been removed by centrifugation and then the de-pectinated samples have been further subjected to fractionation in 4% and 24% KOH solutions for extraction of loosely bound and tightly bound polysaccharides, respectively. This method removes glycoproteins [69]. Amyloid XyG has only been found as a food reserve in plant embryos. Consequently, Amyloid XyG is not believed to interfere with the results obtained from analyses of transgenic roots presented here [7].

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