Alexa-488 dye (Thermo Fisher Scientific) was stably conjugated to the thiol groups of cysteine of the purified EC1236C/44W with a maleimide reagent (Life Technologies) according to the manufacturer’s instructions. This reaction was carried out at 4°C with mixing of Alexa-488 and EC1236C/44W peptide at an equimolar ratio overnight in buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl and 1 mM tris (2-carboxyethyl) phosphine. Both dithiothreitol and β-mercaptoethanol should not be used in the reaction because each of them contains free thiols. Labeled protein was separated from free dye molecules by size exclusion chromatography (Superdex 200). The fluorescently labeled EC1236C/44W (EC12 probe or probe) was aliquoted and frozen in −80°C for in-solution and competitive binding assays.
Maleimide reagent
Manufactured by Thermo Fisher Scientific
Maleimide reagent is a chemical compound used in various applications, such as protein modification and conjugation. It contains a maleimide functional group, which can react with sulfhydryl (thiol) groups in proteins, allowing for the attachment of various molecules or labeling of proteins. The maleimide reagent provides a means for specific and efficient protein labeling and conjugation, making it a useful tool in biochemical and analytical applications.
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Lab products found in correlation
2 protocols using maleimide reagent
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Fluorescent Labeling of EC12 Peptide
2
Fluorescent Labeling of EC12 Peptide
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Alexa-488 dye (Thermo Fisher Scientific) was stably conjugated to the thiol groups of cysteine of the purified EC1236C/44W with a maleimide reagent (Life Technologies) according to the manufacturer’s instructions. This reaction was carried out at 4°C with mixing of Alexa-488 and EC1236C/44W peptide at an equimolar ratio overnight in buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl and 1 mM tris (2-carboxyethyl) phosphine. Both dithiothreitol and β-mercaptoethanol should not be used in the reaction because each of them contains free thiols. Labeled protein was separated from free dye molecules by size exclusion chromatography (Superdex 200). The fluorescently labeled EC1236C/44W (EC12 probe or probe) was aliquoted and frozen in −80°C for in-solution and competitive binding assays.
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