After the click reaction, proteins were precipitated by adding 5 volumes of cold acetone and stored overnight (-20°C). The precipitated proteins were pelleted by centrifugation at 3,500 × g and 4°C for 5 min and washed twice with 500 µL cold methanol. Protein pellets were resuspended in 120 µL PBS containing 1% SDS and desalted by passing through Amicon Ultra 0.5 mL 10K cutoff desalting columns (Merck Millipore) equilibrated with 1% NP-40, 0.1% SDS in PBS. Biotinylated proteins were affinity purified with magnetic streptavidin beads (Thermo Fisher Scientific) (40 μL slurry per sample) at 4°C overnight with slow rotation. Beads were washed twice with 0.2 mL 1% NP-40, 0.1% SDS in PBS for 10 min, three times with 0.2 mL ice-cold 6 M urea in PBS for 15 min, and three times with 0.2 mL ice-cold PBS for 15 min, all at 4 °C with slow rotation. Finally, the beads were rinsed with 50 mM ammonia bicarbonate.
Amicon ultra 0.5 ml 10k cutoff desalting columns
Manufactured by Merck Group
The Amicon Ultra 0.5 mL 10K cutoff desalting columns are a type of laboratory equipment used for the rapid desalting and buffer exchange of protein samples. These columns feature a 10,000 Dalton molecular weight cutoff membrane that allows the removal of small molecules, salts, and other unwanted components from the sample, while retaining the larger protein molecules. The columns have a capacity of 0.5 mL and are designed for convenient, single-use application.
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2 protocols using amicon ultra 0.5 ml 10k cutoff desalting columns
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Biotinylated Protein Purification Protocol
2
Biotinylated Protein Purification Protocol
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After the click reaction, proteins were precipitated by adding 5 volumes of cold acetone and stored overnight (-20°C). The precipitated proteins were pelleted by centrifugation at 3,500 × g and 4°C for 5 min and washed twice with 500 µL cold methanol. Protein pellets were resuspended in 120 µL PBS containing 1% SDS and desalted by passing through Amicon Ultra 0.5 mL 10K cutoff desalting columns (Merck Millipore) equilibrated with 1% NP-40, 0.1% SDS in PBS. Biotinylated proteins were affinity purified with magnetic streptavidin beads (Thermo Fisher Scientific) (40 μL slurry per sample) at 4°C overnight with slow rotation. Beads were washed twice with 0.2 mL 1% NP-40, 0.1% SDS in PBS for 10 min, three times with 0.2 mL ice-cold 6 M urea in PBS for 15 min, and three times with 0.2 mL ice-cold PBS for 15 min, all at 4 °C with slow rotation. Finally, the beads were rinsed with 50 mM ammonia bicarbonate.
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