After incubating the enzyme with the test chemical, 10-mM dithiothreitol and × 4 Laemmli sample loading buffer (Bio-Rad, CA, USA) were added to the reaction mixture. The sample (60-ng protein/lane) and Precision Plus Protein Dual Color Standard (Bio-Rad) were then applied to an SDS-polyacrylamide gel (SDS-PAGE, 10% acrylamide) and then migrated by electrophoresis. The proteins in gels were blotted onto a PVDF membrane (Immobilon-P, Merck), and quinones on the membrane were stained using NBT (0.24 mM) in 2-M glycine-NaOH (pH 10) as described previously [19 (link),20 (link)]. The same samples were separated using a gel, and the proteins in the gel were stained using Flamingo Stain (Bio-Rad) according to the manufactures’ protocol. Detection was performed using a LAS1000 plus (Fujifilm, Tokyo, Japan). This experiment was performed twice.
Flamingo stain
Manufactured by Bio-Rad
Flamingo Stain is a dye used in biological laboratories for the detection and visualization of proteins in polyacrylamide gels. It is a sensitive staining method that can detect nanogram quantities of proteins. The stain binds to the basic amino acid residues in proteins, resulting in the proteins appearing as bright pink bands on the gel.
Automatically generated - may contain errors
2 protocols using flamingo stain
1
Analysis of quinone-modified proteins
2
Quinone Detection in Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
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After incubating the enzyme with the test chemical, 10-mM dithiothreitol and ×4
Laemmli sample loading buffer (Bio-Rad, CA, USA) were added to the reaction mixture.
The sample (60-ng protein/lane) and Precision Plus Protein Dual Color Standard (Bio-Rad) were then applied to an SDS-polyacrylamide gel (SDS-PAGE, 10% acrylamide) and then migrated by electrophoresis. The proteins in gels were blotted onto a PVDF membrane (Immobilon-P, Merck), and quinones on the membrane were stained using NBT (0.24 mM) in 2-M glycine-NaOH (pH 10) as described previously [19, 20] (link). The same samples were separated using a gel, and the proteins in the gel were stained using Flamingo Stain (Bio-Rad) according to the manufactures' protocol. Detection was performed using a LAS1000 plus (Fujifilm, Tokyo, Japan). This experiment was performed twice.
Laemmli sample loading buffer (Bio-Rad, CA, USA) were added to the reaction mixture.
The sample (60-ng protein/lane) and Precision Plus Protein Dual Color Standard (Bio-Rad) were then applied to an SDS-polyacrylamide gel (SDS-PAGE, 10% acrylamide) and then migrated by electrophoresis. The proteins in gels were blotted onto a PVDF membrane (Immobilon-P, Merck), and quinones on the membrane were stained using NBT (0.24 mM) in 2-M glycine-NaOH (pH 10) as described previously [19, 20] (link). The same samples were separated using a gel, and the proteins in the gel were stained using Flamingo Stain (Bio-Rad) according to the manufactures' protocol. Detection was performed using a LAS1000 plus (Fujifilm, Tokyo, Japan). This experiment was performed twice.
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