Activity staining on polyacrylamide gels of trypsin inhibitors from leaves of sweet potato (Ipomoea batatas L. Lam) varieties

Abstract

The failure of activity staining of trypsin inhibitors in crude leaf extracts of sweet potato varieties including Tainong 27, Tainong 34, and Tainong 57 on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel was prevented by dipping the gels in solutions containing 10-40 mM hydrogen peroxide, 10 mM Tris buffer (pH 7.9) for 30 min before staining.

Detection of protease activities using specific aminoacyl or peptidyl p ‐nitroanilides after sodium dodecyl sulfate — polyacrylamide gel electrophoresis and its applications

“…The activity staining of trypsin inhibitors showed a clear zone against a magenta background. Although APNE was frequently used as a substrate for trypsin inhibitor activity staining [21,29], BANA seemed more suitable than APNE concerning far as substrate specificity.…”

Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels

“…One was fixed with 12.5% trichloroacetic acid and then stained for proteins with Coomassie Brilliant Blue R-250 dye [24]. The other part was immersed and shaken for 10 min twice in 25% v/v isopropanol in 10 mM Tris-HCl buffer (pH 7.9) to remove the SDS [19,[25][26][27][28], and then finally equilibrated for renaturation in 50 mM Tris-HCl buffer (pH 7.9) for 15 min before GR activity staining. For native PAGE gels, one part was fixed with 12.5% trichloroacetic acid and then stained for proteins [24], while the other part was dipped twice for 15 min in each 50 mM Tris-HCl buffer (pH 7.9) before GR activity staining.…”

“…The absorbed GR was eluted by 2 mM NADP 1 in 100 mM Tris-HCl buffer (pH 7.9) containing 100 mM NaCl. Using 25% v/v isopropanol in 10 mM Tris-HCl buffer (pH 7.9) to remove SDS and then finally equilibrating for renaturation in 50 mM TrisHCl buffer (pH 7.9) for 15 min has been successfully reported for several enzyme stains [19,[25][26][27][28]. The mixture of DTNB 1 GSSG 1 NADPH was used as a substrate, and then gels were stained negatively by MTT/ PMS.…”

Selection of Sweetpotato Lines with High Protein Content and/or Low Trypsin Inhibitor Activity

“…TrisGlycine gels (gel concentration of 14%) were used with Tris-Glycine running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS, pH 8.3). Samples for this system were prepared according to the method of Hou and Lin (1998), where the reducing agent was not added to the samples, and binding of the samples with SDS was performed at room temperature overnight instead of heating. Reduced samples were prepared by the addition of 50 mM of dithiothreitol (DTT) and heating at 70°C for 10 min.…”

“…Reduced samples were prepared by the addition of 50 mM of dithiothreitol (DTT) and heating at 70°C for 10 min. TI activity staining of the nonreduced TI bands was carried out according to the method of Hou and Lin (1998). NuPAGE gels (gel concentration of 10%) were used with NuPAGE MES (2-(N-morpholino) ethane sulfonic acid) running buffer (50 mM MES, 50 mM Tris, 3.5 mM SDS, 1 mM EDTA, pH 7.3).…”