Purified ScYop1p was mixed with Alexa Fluor 647 NHS ester dye (Thermo Fischer Scientific) at a 1:1 protein:dye molar ratio and incubated at room temperature for 1 hr. The labeling reaction was quenched by addition of 100 mM Tris pH 8.0. After an incubation of 1 hr at 4 °C, excess dye was removed by passing the sample over Sephadex G-50 resin (GE healthcare). The labeled protein was collected and concentrated by ultrafiltration using a 50,000 MW cut-off filter (Amicon Ultra, EMD Millipore). The labeling efficiency was ~50%, as calculated by comparing the absorbance of the protein at 280 nm and the absorbance of the dye using the pre-configured “Proteins & Labels” function on a Nano Drop 2000c Spectrophotometer (Thermo Scientific).
Alexa fluor 647 nhs ester dye
Manufactured by Thermo Fisher Scientific
Alexa Fluor 647 NHS ester is a fluorescent dye used for labeling proteins and other biomolecules. It has an excitation maximum at 650 nm and an emission maximum at 665 nm, making it suitable for detection in the far-red/near-infrared region of the spectrum.
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2 protocols using alexa fluor 647 nhs ester dye
1
Fluorescent Labeling of ScYop1p Protein
2
Fluorescent Labeling of ScYop1p Protein
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Purified ScYop1p was mixed with Alexa Fluor 647 NHS ester dye (Thermo Fischer Scientific) at a 1:1 protein:dye molar ratio and incubated at room temperature for 1 hr. The labeling reaction was quenched by addition of 100 mM Tris pH 8.0. After an incubation of 1 hr at 4 °C, excess dye was removed by passing the sample over Sephadex G-50 resin (GE healthcare). The labeled protein was collected and concentrated by ultrafiltration using a 50,000 MW cut-off filter (Amicon Ultra, EMD Millipore). The labeling efficiency was ~50%, as calculated by comparing the absorbance of the protein at 280 nm and the absorbance of the dye using the pre-configured “Proteins & Labels” function on a Nano Drop 2000c Spectrophotometer (Thermo Scientific).
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