Gibberellic acid and benzyladenine plant growth regulators (purchased from Sigma Company) at three levels of 0, 100 and 200 mg/l were prepared and sprayed on plant shoots in three stages with 15-day intervals17 . Tween 20 (0.1%) (purchased from Sigma Company) was used in the solution to enhance its longevity and uptake. Hormone spraying was done inthe early hours of the morning using a sprayer on shoots and leaves of dwarf schefflera. Control plants were sprayed with distilled water.
Tween 20 0.1
Manufactured by Merck Group
Sourced in France
Tween 20 (0.1%) is a non-ionic surfactant commonly used in laboratory procedures. It is a polyoxyethylene sorbitan monolaurate solution with a concentration of 0.1%.
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4 protocols using tween 20 0.1
1
Gibberellic Acid and Benzyladenine Spray on Dwarf Schefflera
2
Western Blot Protein Detection Protocol
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Proteins were extracted with RIPA buffer (Tris-HCl 50 mM pH7.4, NaCl 150 mM, NP-40 1%, sodium deoxycholate 0.25%, sodium orthovanadate 1 mM) complemented with protease and protease/phosphatase inhibitors (Roche, Basel, Switzerland). Cell lysates were sonicated for 15 sec and centrifuged. Proteins were then measured using Bradford protein assay. 30 μg protein were loaded on SDS-polyacrylamide gels 7.5% and transferred onto a PVDF membrane (Merck). After transfer, membranes were blocked for 1 h in Tris-Buffered Saline (TBS) with Tween-20 0.1% (Sigma) and bovine serum albumin (BSA) 5% or milk 5% and then incubated overnight at 4°C with primary antibody (Table 2 ) in TBS-BSA 5% or milk 5%. Then, membranes were incubated with anti-rabbit (1:2000, Enzo Life Sciences, Plymouth Meeting, PA, USA) or anti-mouse (1:1000, Cell Signaling Technology, Danvers, Massachusetts, USA) secondary antibodies, and proteins were detected using chemiluminescence (Super Signal West Pico Plus, Thermo Fisher Scientific) and Fusion Solo S equipment (Vilber Lourmat, Collegien, France).
3
Leptin's Effects on Mammary Cells
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Mammary epithelial cells (HMEC, MCF7 and MDA-MB-231), synchronized in serum free medium for 24 h before initiation of leptin treatment, grew for 0, 1, 6 or 24 h in their media either with or without recombinant human leptin (R&D, Abingdon, United Kingdom) at physiological (10 ng/ml) or obese (100 ng/ml) concentrations.
Cells were harvested after trypsinization and three phosphate buffer saline washes. Total cell lysates were obtained by two successive thawing-freezing cycles in TrisHCl 25 mM buffer pH 7.4 containing Tween 20 0.1% (Sigma-Aldrich, Saint-Quentin-Fallavier, France), with 15-s periods in an ultra-sound bath, and then stored at − 80 °C until analysis.
Proteins were quantified by the bicinchonic acid method (Interchim, Montluçon, France) using a standard curve with a bovine serum albumin solution (2 g/l), according to the manufacturer’s instructions.
Cells were harvested after trypsinization and three phosphate buffer saline washes. Total cell lysates were obtained by two successive thawing-freezing cycles in TrisHCl 25 mM buffer pH 7.4 containing Tween 20 0.1% (Sigma-Aldrich, Saint-Quentin-Fallavier, France), with 15-s periods in an ultra-sound bath, and then stored at − 80 °C until analysis.
Proteins were quantified by the bicinchonic acid method (Interchim, Montluçon, France) using a standard curve with a bovine serum albumin solution (2 g/l), according to the manufacturer’s instructions.
4
Western Blot Analysis of Cell Samples
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In protein sample preparation, the supernatant of cell culture was collected and the cells were lysed in ice-cold RIPA lysis buffer (Beyotime) with EDTA-free Protease Inhibitor Cocktail Tablets (Roche). The protein samples were separated on a 10% SDS-PAGE or non-reducing SDS-PAGE and electrophoretically transferred to a PVDF membrane (Millipore, Billerica, MA). After they were blocked in Tris-buffered saline (pH 7.4) containing Tween-20 (0.1%) (Sigma-Aldrich) with 5% (w/v) nonfat milk, the membranes were incubated in 5% milk with the antibodies specific for the proteins (rabbit anti-IL-12 A mAb, 1:2000, anti-IL-12B mAb, 1:2000,anti-cRel mAb,1:2000, Boster, Wuhan, China; rabbit anti-STAT3 mAb, 1:1000, anti-pSTAT3 mAb, 1:1000,CST,USA; mouse anti-β-Tubulin mAb, 1:4000, Sungene Biotech, Tianjin, China; mouse anti-GAPDH mAb, 1:2000, anti-PCNA mAb, 1:2000, Abbkine Scientific,USA), separately, overnight at 4 °C. After that, the membranes were exposed to horseradish peroxidase-conjugated goat anti-rabbit/mouse secondary antibody (1:5000, Servicebio, Wuhan, China) for 1 h at room temperature. Finally, the signals were detected using an ECL kit (MCE) according to the manufacturer’s instructions.
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