Initially, Protein Deglycosylation Mix II (New England BioLabs), which contains a mixture of different enzymes, was used unsuccessfully to remove the glycosylation from the LAL14/1 pili. Subsequently, deglycosylation of LAL14/1 pili was performed using the Glycoprofile IV™ chemical deglycosylation kit (Sigma Aldrich). Briefly, 50 μL of concentrated pili were lyophilized in a small glass tube. Then, 150 μL of Trifluoromethansulfonic acid (TFMS) was added to the tube and the mixture was incubated at 4 °C for 1 hour. Following this, 150 μL of 60 % pyridine solution, cooled to ~15 °C with a methanol-dry ice bath, was added drop-wise to the reaction tube, neutralizing the TFMS acid. Using a 2000 molecular mass cut off Slide-A-Lyzer® dialysis cassete (Thermo Scientific), the reaction solutions were removed from the deglycosylated pili sample with overnight dialysis at 4 °C into TRIS/HCl pH 7.8 buffer. After dialysis, small amounts of aggregates, presumably deglycosylated pili, were observed in the cassette. Centrifugation for 15 min at 4 °C and 20,000 RCF was used to pellet the aggregates. The pellet was re-suspended with 50 μL of tris buffer pH 7.8.
Glycoprofile 4 chemical deglycosylation kit
Manufactured by Merck Group
Glycoprofile IV™ is a chemical deglycosylation kit developed by Merck Group. The kit is designed to remove glycans from glycoproteins, enabling the analysis of the underlying protein structure.
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Lab products found in correlation
5 protocols using glycoprofile 4 chemical deglycosylation kit
1
Chemical Deglycosylation of LAL14/1 Pili
2
Coccolith Isolation and Deglycosylation Protocol
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Sample preparation is described briefly here, and in detail in Supplementary Note 5 . Cells for all experiments were harvested at early–mid log phase (2–4 × 106 cells/mL). Cultures for total protein extraction (quantitative experiments) were sonicated in an SDS extraction buffer followed by centrifugation to remove cell debris. Coccoliths and ESOM were isolated as described above. This material was deglycosylated with Trifluoromethanesulfonic acid (TFMS) on ice according to the manual of the GlycoProfile™ IV Chemical Deglycosylation Kit (Sigma-Aldrich). Coccolith vesicles were enriched from recalcifying cells which had not yet exocytosed their first coccolith. After disruption using a French Press, immature coccoliths were enriched by centrifugation through a sucrose step gradient. Samples were prepared for mass spectrometry using an on-filter digest protocol followed by desalting using C18 columns. TMT labeling was performed as described in Supplementary Note 5 .
3
Chemical Deglycosylation of LAL14/1 Pili
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Initially, Protein Deglycosylation Mix II (New England BioLabs), which contains a mixture of different enzymes, was used unsuccessfully to remove the glycosylation from the LAL14/1 pili. Subsequently, deglycosylation of LAL14/1 pili was performed using the Glycoprofile IV™ chemical deglycosylation kit (Sigma Aldrich). Briefly, 50 μL of concentrated pili were lyophilized in a small glass tube. Then, 150 μL of Trifluoromethansulfonic acid (TFMS) was added to the tube and the mixture was incubated at 4 °C for 1 hour. Following this, 150 μL of 60 % pyridine solution, cooled to ~15 °C with a methanol-dry ice bath, was added drop-wise to the reaction tube, neutralizing the TFMS acid. Using a 2000 molecular mass cut off Slide-A-Lyzer® dialysis cassete (Thermo Scientific), the reaction solutions were removed from the deglycosylated pili sample with overnight dialysis at 4 °C into TRIS/HCl pH 7.8 buffer. After dialysis, small amounts of aggregates, presumably deglycosylated pili, were observed in the cassette. Centrifugation for 15 min at 4 °C and 20,000 RCF was used to pellet the aggregates. The pellet was re-suspended with 50 μL of tris buffer pH 7.8.
4
Deglycosylation of Bl-Eng2 Protein
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Deglycosylation of Bl-Eng2 was performed by using the GlycoProfile™ IV Chemical Deglycosylation kit (Sigma-Aldrich) according to the manufacturer’s specifications. Treatment with trifluormethanosulfonic acid (TFMS) was used to remove N- and O-glycans from Bl-Eng2 while preserving the protein backbone [34 (link)]. PNGase F (New England Biolabs) digestion was employed to remove N-linked oligosaccharides from Bl-Eng2, and Proteinase K (Promega) was used to digest the protein back-bone. Pro-Q™ Emerald 300 glycoprotein gel stain kit (Invitrogen) was used to detect the presence of sugar in Bl-Eng2 before and after deglycosylation. Colloidal Coomassie blue staining (Invitrogen) or silver stain was used to evaluate protein homogeneity and migration in SDS-PAGE.
5
Deglycosylation of Cryptosporidium parvum Mucin-Like Glycoprotein
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