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11 protocols using WESTSAVE

To extract total proteins, the middle parts of the first leaves in the main culms of 6-week-old rice plants grown under LD conditions (14 h light/10 h dark) were used. To extract total proteins, leaf tissues were ground in liquid nitrogen and 10-mg aliquots were homogenized with 100 µl of sample buffer [50 mM Tris, pH 6.8, 2 mM EDTA, 10% glycerol, 2% sodium dodecyl sulfate (SDS), and 6% 2-mercaptoethanol]. The homogenates were centrifuged at 10000 g for 3 min, and the supernatants were denatured at 80 °C for 5 min. A 4-µl aliquot of each sample was subjected to 12% (w/v) polyacrylamide SDS-polyacrylamide gel electrophoresis (PAGE), and the resolved proteins were electroblotted onto a Hybond-P membrane (GE Healthcare, USA). Antibodies against photosystem proteins Lhca1, Lhca2, Lhcb1, Lhcb2, Lhcb4, CP43, and PsaA (Agrisera, Sweden) were used for immunoblot analysis. The level of each protein was measured using the ECL system with WESTSAVE (AbFrontier, Korea) according to the manufacturer’s protocol.
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Cells harvested on the 4, 9, and 14 days of differentiation were lysed on ice for 20 min with a radioimmunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor cocktail (GenDEPOT, Harris County, TX, USA). The lysates were centrifuged at 12,000 rpm for 30 min at 4 °C and transferred to a new 1.5 mL tube. The protein concentrations were measured using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were incubated with 5% skim milk at room temperature for 1 h, then incubated overnight at 4 °C with primary antibodies, anti-alpha-SMA (Abcam, Cambridge, UK), anti-CNN1 (Abcam), and anti-SM-MHC (Abcam). After washing extensively, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 2 h. The signal was detected using WESTSAVE (AbFrontier, Korea) and an enhanced chemiluminescence system. ImageJ (https://imagej.nih.gov/ij/,download.html, accessed on 14 September 2021) software was used to quantify the band intensity of the Western blot.
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The whole-cell lysate was prepared using Radioimmunoprecipitation assay buffer (RIPA buffer) prepared in 50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, protease inhibitor cocktail, and phosphatase inhibitor cocktail. Protein assays were carried out to normalize the proteins using a Bradford protein assay (Thermo Scientific, Waltham, MA, USA). Proteins were resolved by SDS-PAGE and were transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Burlington, MA, USA). Membranes were blocked in 5% bovine serum albumin (BSA) for 1 h at rt and incubated with indicated antibodies overnight at 4 °C. Membranes were washed in Tris-buffered saline, 0.1% Tween 20 (TBST) for 1 h at rt and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at rt. Membranes were washed five times with TBST, and chemiluminescence was detected using Westsave™ (Abfrontier, Seoul, Korea). Gels were imaged using FUSION-Solo.4.WL (Vilber Lourmat, Collégien, France).
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Protein extracts were prepared from rosette leaves of Arabidopsis thaliana. A 10mg aliquot of leaf tissue was ground in liquid nitrogen and homogenized with 100 μl of sample buffer [50mM TRIS–HCl, pH 6.8, 2mM EDTA, 10% (w/v) glycerol, 2% SDS, and 6% 2-mercaptoethanol] was used to suspend the protein extracts. The protein samples were subjected to SDS-PAGE. Gels were stained with Coomassie Brilliant Blue R-250 (Sigma–Aldrich). Antibodies against photosynthetic proteins, including Lhca3, Lhcb1, Lhcb2, Lhcb4, Lhcb5, CP43, D1, and PsaA (Agrisera, Sweden), were used for immunoblot analysis. Each protein was detected using an electrochemiluminescence (ECL) system (WESTSAVE, AbFRONTIER, Seoul, Korea) according to the manufacturer’s manual.
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HepG2 cells (1 × 105 cells/well) were seeded into six-well plates, and the cells were incubated with vehicle (control) or 40 µM DPHC for 1 h, and then further incubated with or without 0.4 mM palmitate for 24 h. The cells were lysed using 1% Triton X-100-PBS and protease inhibitor cocktail (GenDEPOT, Barker, TX, USA) for 20 min, on ice. The lysates were fractionated by centrifugation at 12,000 rpm for 20 min at 4 °C, and the pellets were used for western blotting. Protein concentrations were measured using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). The lysates were separated by SDS-PAGE and transferred to PVDF (polyvinylidene fluoride) membranes (Millipore, Billerica, MA, USA). Membranes were incubated with 5% skimmed milk for 1 h at room temperature, and then incubated with primary antibodies overnight at 4 °C. After washing extensively, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). Signals were detected using WESTSAVE (Ab Frontier, Seoul, Korea) and an enhanced chemiluminescence system. ImageJ software was used to quantify the band intensities of western blots. The primary antibodies used were anti-SREBP1, anti-C/EBPβ, anti-ChREBP, anti-FAS, and anti-β-actin. All primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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Total protein extracts were prepared from leaf tissues using the middle section of the third leaf in the main culm of 2-month-old rice plants grown in the growth chamber under LD conditions. Leaf tissues were ground in liquid nitrogen, and 10-mg aliquots were homogenized with 100 μl of sample buffer (50 mM Tris pH 6.8, 2 mM EDTA, 10% glycerol, 2% SDS, and 6% β-mercaptoethanol). The homogenates were centrifuged at 10 000 g for 3 min, and the supernatants were denatured at 80 °C for 5 min. A 4-μl aliquot of each sample was subjected to 12% (w/v) SDS-PAGE, followed by electroblotting onto a Hybond-P membrane (GE Healthcare). Antibodies against photosystem proteins, including Lhca1, Lhca2, Lhcb1, Lhcb2, and CP43, were used for immunoblot analysis, and RbcL was visualized by Coomassie Brilliant Blue (CBB) staining. The level of each protein was examined using the ECL system with WESTSAVE (AbFRONTIER, Korea) according to the manufacturer’s protocol.
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Photosynthesis-related proteins were detected as previously described, with some modifications (Han et al. 2012 (link); Kwon et al. 2015 (link); Kwon et al. 2017 (link)). Leaf tissue (10 mg) was homogenized with 100 μL of SDS-PAGE sample buffer [50 mM Tris pH 6.8, 2 mM EDTA, 10% (w/v) glycerol, 2% SDS, and 6% 2-mercaptoethanol] and denatured at 100 °C for 5 min, then samples were subjected to SDS-PAGE. For immunoblot analysis, 5 μL of each protein sample was used. The resolved proteins were electroblotted onto the Immobilon-P Transfer Membrane (Millipore). Antibodies against photosystem proteins (Lhca2, Lhcb2, Lhcb4, and D1) were obtained from Agrisera (Sweden). To detect the horseradish peroxidase activity of secondary Antibodies (Sigma), an ECL detection kit, WESTSAVE (AbFRONTIER, Korea), and a chemiluminescence system (FUSION-FX7, France) were used according to the manufacturers’ protocols. The Rubisco large subunit (RbcL) was visualized by staining with Coomassie brilliant blue reagent (Sigma-Aldrich) after immunoblot analysis.
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HEK cells (4 × 106 cells/well) were seeded onto 6-well plates, and the cells were incubated with vehicle (control) or 40 μM DPHC for 1 h and then further incubated with or without 1 mM MGO for 24 h. The cells were lysed using 1% Triton X-100-PBS and protease inhibitor cocktail (GenDEPOT, Barker, TX) for 20 min on ice. The lysates were fractionated by centrifugation at 12,000 rpm for 20 min at 4°C. The supernatant was used as the cytosolic fraction, and the pellets were used as the nuclear fraction for Western blotting. The protein concentrations were measured using a DC protein assay kit (Bio-Rad, Hercules, CA). The lysates were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA). Membranes were incubated with 5% skimmed milk for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C. After washing extensively, membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA). The signal was detected using WESTSAVE (AbFrontier, Korea) and the enhanced chemiluminescence system. ImageJ software was used to quantify the band intensity of Western blot. Primary antibodies used were anti-Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-lamin B (Santa Cruz Biotechnology, Santa Cruz, CA).
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Total protein extracts were prepared from leaf tissues using detached leaf discs from 1-month-old rice plants before and after 2 or 4 d of dark treatment. Leaf tissues were ground in liquid nitrogen, and 10-mg aliquots were homogenized with 100 mL of sample buffer (50 mM Tris, pH 6.8, 2 mM EDTA, 10% [v/v] glycerol, 2% [w/v] SDS, and 6% [v/v] 2-mercaptoethanol). The homogenates were centrifuged at 10,000g for 3 min, and the supernatants were denatured at 80°C for 5 min. A 4-mL aliquot of each sample was subjected to 12% (w/v) SDS-PAGE, followed by electroblotting onto a Hybond P membrane (GE Healthcare). Antibodies against photosystem proteins, including Lhca1 (Agrisera, no. AS01005), Lhcb2 (Agrisera, no. AS01003), Lhcb4 (Agrisera, no. AS04045), PsaA (Agrisera, no. AS06172), CP43 (Agrisera, no. AS0111787), and a-tubulin (Agrisera, no. AS10680), and anti-rabbit IgG horseradish peroxidase-linked antibody (Cell Signaling Technology, no. 7074) were used for immunoblot analysis, and RbcL was visualized by Coomassie Brilliant Blue staining. The level of each protein was examined using the enhanced chemiluminescence system with WESTSAVE (AbFRONTIER) according to the manufacturer's protocol.
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Total protein extracts were prepared from leaf tissues, using the middle section of the third leaf in the main culm of each 2-month-old rice plant grown under LD. Leaf tissues were ground in liquid nitrogen, and 10 mg aliquots were homogenized with 100 μl of sample buffer (50 mM Tris, pH 6.8, 2 mM EDTA, 10% glycerol, 2% SDS, and 6% 2-mercaptoethanol). The homogenates were centrifuged at 10,000 × g for 3 min, and the supernatants were denatured at 80°C for 5 min. A 4 μl aliquot of each sample was subjected to 12% (w/v) SDS-PAGE, followed by electroblotting onto a Hybond-p membrane (GE Healthcare). Antibodies against photosystem proteins Lhca1, Lhca2, Lhcb1, Lhcb2, Lhcb4, Lhcb5, CP43, and PsaA (Agrisera, Sweden) were used for immunoblot analysis, and RbcL was visualized by Coomassie Brilliant Blue (CBB) staining. The level of each protein was examined using the ECL system with WESTSAVE (AbFRONTIER, Korea) according to the manufacturer’s protocol.
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