Protein Deglycosylation Mix II (NEB) was used for deglycosylation reaction following manufacturer's instructions. Briefly, 2 μg proteins were mixed with Deglycosylation Mix Buffer 1 (non-denaturing reaction) or Buffer 2 (denaturing reaction, with an additional incubation at 75 °C for 10 min), ...
First, to increase proteomic coverage of glycoproteins, crosslinked virion samples were deglycosylated using Protein Deglycosylation Mix II (P6044, NEB) under denaturing conditions according to manufacturer instructions. Lysis of virion samples was performed by adding 3 volumes of lysis buffer containing ...
i.e., glycosylation and phosphorylation, were responsible for the observed MTALTND4 band (~ 24 kDa) at higher MW than expected (11.5 kDa), whole cell lysates were treated with a complete protein deglycosylation mix II kit (NEB, Ipswich, MA, USA; Cat# P6044) following the manufacturer’...
(as required for the IHC protocol using 1E6 antibody40) and incubated with a deglycosylation mix composed of: protein deglycosylation mix II, beta 1-3 galactosidase and alpha 1-3, 4, 6 galactosidase (New England Biolabs (NEB), P6044Sm P0726S and P0747S respectively), at RT for 30 ...
Deglycosylation Of each SAPN, 15.5 μg was brought up to 49 μL in the respective refolding buffer. One microliter of Protein Deglycosylation Mix II (New England Biosciences, Ipswich, MA) was added to the samples. Reactions were held at 25 °C for 30 minutes and proteins were identified on...
LCP/oMstS-/tMranSsblaytiPoonoa-l Preparation of sample for LC/MS/MS analysis for glycosylation. 25 μg of protein samples were digested by trypsin either (a) without treatment or (b) upon treatment with deglycosylation Mix II [Protein Deglycosylation Mix II (P6044S, Biolabs)] ...
Magnetic chitin beads (NEB) were then used to remove the PNGase F, which contains a chitin-binding domain (CBD). 50 μL of chitin beads were washed twice with 200 μL of selection buffer, and then combined with the deglycosylation reaction. The mixture was incubated for 30 min on...
Deglycosylation assay Being all the salivary proteins secreted, we expect that they might be glycosylated. As the majority of glycosylation is N-glycan in the majority of insect species analyzed,81 we treated extracts with recombinant PNGase-F (New England BioLabs). Briefly, Glycoprotein Denaturing ...
Pellet was washed with 10 mM Tris, dissolved in Glycoprotein denaturation buffer (NEB) and heated for 5 min at 80 °C. Protein concentration was measured using the RC/DC Kit from Bio-Rad and adjusted to 1.5 µg/µl. A deglycosylation assay was performed using the PNGase-F ...
Currently, licensed influenza virus vaccines are designed and tested only for their ability to elicit hemagglutinin (HA)-reactive, neutralizing antibodies. Despite this, the purification process in vaccine manufacturing often does not completely remove o