Author: Makadiya, Niraj; Brownlie, Robert; van den Hurk, Jan; Berube, Nathalie; Allan, Brenda; Gerdts, Volker; Zakhartchouk, Alexander
Title: S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen Document date: 2016_4_1
ID: 1r3doeic_40
Snippet: PNGase F and O-Glycosidase was purchased from NEB. Briefly, 2 μg of purified S1 protein was added with 1 μL of 10X glycoprotein denaturing buffer (NEB) in total of 10 μL of reaction volume and denatured at 95°C for 5 min. Then, the mixture was chilled on ice for 30 s followed by centrifugation for 10 s at 10,000 X g. Reaction volume was increased to 20 μL by adding 2 μL 10X GlycoBuffer (NEB), 2 μL 10 % NP40, water and 1 μL of enzyme PNGas.....
Document: PNGase F and O-Glycosidase was purchased from NEB. Briefly, 2 μg of purified S1 protein was added with 1 μL of 10X glycoprotein denaturing buffer (NEB) in total of 10 μL of reaction volume and denatured at 95°C for 5 min. Then, the mixture was chilled on ice for 30 s followed by centrifugation for 10 s at 10,000 X g. Reaction volume was increased to 20 μL by adding 2 μL 10X GlycoBuffer (NEB), 2 μL 10 % NP40, water and 1 μL of enzyme PNGase F or 2 μL of O-glycosidase and incubated at 37°C for 1 h. The extent of glycosylation was analyzed by mobility shift on SDS-PAGE followed by Western blotting.
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