Young watermelon fruits (~10 DAP) were sliced thinly using a razor blade, and the sections were incubated in the wall digesting solution (500 mM mannitol (Thermo Fisher Scientific, Waltham, MA), 10 mM CaCl2, 20 mM KCl, 20 mM MES-KOH (Thermo Fisher Scientific, Waltham, MA), 1% BSA, 1% Cellulase Onozuka (Yakult, Tokyo, Japan), 0.5% Marcerozyme 10 (Yakult, Tokyo, Japan), and 0.1% β-mercaptoethanol at pH 5.6 (TCI America, Portland, Oregon) for 2.5 h. The protoplasts were filtered through a 70-μm mesh and transformed with the GFP fusion constructs, as described in [49 (link)].
β mercaptoethanol
Manufactured by Tokyo Chemical Industry
Sourced in Japan
β-mercaptoethanol is a clear, colorless liquid with a characteristic odor. It is commonly used as a reducing agent in various biochemical applications, such as protein extraction, purification, and stabilization.
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Lab products found in correlation
3 protocols using β mercaptoethanol
1
Isolation and Transformation of Watermelon Protoplasts
2
SDS-PAGE Analysis of Protein Filaments
3
SDS-PAGE Analysis of Protein Filaments
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To separate via sodium-dodecyl sulfate -polyacrylamide gel electrophoretic (SDS-PAGE), the filament samples were mixed with reducing sample buffer with 0.3% w/v final concentration of SDS and 1.5% v/v final concentration of β-mercaptoethanol (TCI America). All samples were heated for 5 min at 100 °C and then cooled and spun at 2500xg for 1 min before loading samples into the gel. The filament samples were run on 4-16% Tricine-SDS-PAGE gels 89 prepared using Acryl/Bis (37.5:1, 40% w/v) solution (VWR) with an initial voltage of 30 V for 20 min and next voltage step of 200V for 3 hours. Spectra multicolor broad range protein ladder (ThermoFisher) was used to reference the molecular weight of the protein bands. Gels were stained both with silver stain 90 or with 3,3′,5,5′-tetramethylbenzidine (TMB) 91 after washing with ultrapure water. βmercaptoethanol addition was omitted for Heme (TMB) stain samples.
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