The Alexa-546 C5-malemide dyes (Invitrogen) were mixed at a 1:1 ration with purified zinc-bound C146S Sod1 in a buffer containing 20 mM Tris pH 7.5, 300 mM NaCl and 0.5 mM TCEP. The mixture is allowed to incubate in the dark at 4°C overnight. The amount of protein is not critical, but the concentration of the protein is kept near 1–5 μM to avoid aggregation events. The next morning the protein is concentrated in spin concentrators to both lower the total volume and remove some of the excess dye. The sample is then loaded onto a Superdex 200 SEC column from GE. The column is wrapped in foil to keep the sample in the dark during the separation. The sample is fractionated and then run on SDS-PAGE. The excess dye elutes near the bed volume of the column and the protein sample is run on UV-vis at 280 nm to find the final concentration.
Alexa 546 c5 malemide dyes
Manufactured by Thermo Fisher Scientific
Alexa-546 C5-maleimide dyes are a type of fluorescent labeling reagent used in various biological and biochemical applications. These dyes contain a maleimide reactive group that can covalently attach to sulfhydryl (thiol) groups, allowing for the labeling of proteins, peptides, and other biomolecules. The Alexa-546 dye exhibits absorption and emission maxima at approximately 554 nm and 571 nm, respectively, making it suitable for detection and visualization using standard fluorescence techniques.
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Lab products found in correlation
2 protocols using alexa 546 c5 malemide dyes
1
Labeling Sod1 Protein with Alexa Dyes
2
Labeling Sod1 Protein with Alexa Dyes
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The Alexa-546 C5-malemide dyes (Invitrogen) were mixed at a 1:1 ration with purified zinc-bound C146S Sod1 in a buffer containing 20 mM Tris pH 7.5, 300 mM NaCl and 0.5 mM TCEP. The mixture is allowed to incubate in the dark at 4°C overnight. The amount of protein is not critical, but the concentration of the protein is kept near 1–5 μM to avoid aggregation events. The next morning the protein is concentrated in spin concentrators to both lower the total volume and remove some of the excess dye. The sample is then loaded onto a Superdex 200 SEC column from GE. The column is wrapped in foil to keep the sample in the dark during the separation. The sample is fractionated and then run on SDS-PAGE. The excess dye elutes near the bed volume of the column and the protein sample is run on UV-vis at 280 nm to find the final concentration.
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