Author: Asif Shajahan; Nitin T. Supekar; Anne S. Gleinich; Parastoo Azadi
Title: Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2 Document date: 2020_4_3
ID: f0xsisdg_12
Snippet: We have procured culture supernatants of HEK 293 cells expressing SARS-CoV-2 subunit 1 and subunit 2 separately. The proteins were expressed with a His tag with Val16 to Gln690 for subunit 1 and Met697 to Pro1213 for subunit 2. According to manufacturers, SDS-PAGE of the proteins showed a higher molecular weight than the predicted 75 and 60 kDa, respectively, due to glycosylation. Since the proteins were unpurified, we fractionated them through S.....
Document: We have procured culture supernatants of HEK 293 cells expressing SARS-CoV-2 subunit 1 and subunit 2 separately. The proteins were expressed with a His tag with Val16 to Gln690 for subunit 1 and Met697 to Pro1213 for subunit 2. According to manufacturers, SDS-PAGE of the proteins showed a higher molecular weight than the predicted 75 and 60 kDa, respectively, due to glycosylation. Since the proteins were unpurified, we fractionated them through SDS-PAGE on separate lanes and cut the bands corresponding to subunit 1 and subunit 2. The gels were stained with Coomassie dye, and gel bands were cut into small pieces, de-stained, reduced, alkylated and subjected to in-gel protease digestion. We employed trypsin, chymotrypsin, and both trypsinchymotrypsin in combination to generate glycopeptides that contain a single N-linked glycan site.
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