The glycosylation of TtC was confirmed by glycoprotein staining. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis of glycosylated protein was performed under reducing conditions on the 12 % gel. Glycoprotein staining was conducted using Sigma’s glycopeptide staining kit (GlycoPro) where glycoproteins are stained magenta. This detection system is a modification of periodic acid-Schiff (PAS) method (Zacharius et al. 1969 (link)). After detection of the glycosylated protein, the same gel was stained with Coomassie blue to detect other protein bands. The presence of N-linked glycosylation was further confirmed using the deglycosylating enzyme peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase PNGase F (New England Biolabs).
Peptide n4 n acetyl beta glucosaminyl asparagine amidase pngase f
Manufactured by New England Biolabs
Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase PNGase F is an enzyme that cleaves the linkage between the asparagine residue and the N-acetylglucosamine residue of N-linked glycoproteins. It catalyzes the removal of N-linked oligosaccharides from glycoproteins.
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Lab products found in correlation
2 protocols using peptide n4 n acetyl beta glucosaminyl asparagine amidase pngase f
1
Glycosylation Characterization of TtC Protein
2
Synovial Fluid Protein Digestion
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Performed using the FASP protein digestion
kit (Protein Discovery,
San Diego, CA) according to manufacturer’s instructions using
30 kDa cutoff spin filters. Ninety micrograms of total synovial fluid
protein was digested overnight at 37 °C with 2 μg of sequencing
grade modified trypsin (Promega, Fitchburg, MA). To assess the need
of glycan removal when working with synovial fluid, 500 U peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine
amidase (PNGase F) (New England BioLabs, Ipswich, MA) was added to
these samples prior to the trypsin digestion step, and samples were
incubated overnight at 37 °C, after which the normal FASP protocol
was continued. After trypsin digestion, the samples were desalted
with TARGA C18 columns (Nest Group, Southborough, MA) and resuspended
in 5% acetonitrile (ACN) and 5% formic acid (FA) prior to analysis.
kit (Protein Discovery,
San Diego, CA) according to manufacturer’s instructions using
30 kDa cutoff spin filters. Ninety micrograms of total synovial fluid
protein was digested overnight at 37 °C with 2 μg of sequencing
grade modified trypsin (Promega, Fitchburg, MA). To assess the need
of glycan removal when working with synovial fluid, 500 U peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine
amidase (PNGase F) (New England BioLabs, Ipswich, MA) was added to
these samples prior to the trypsin digestion step, and samples were
incubated overnight at 37 °C, after which the normal FASP protocol
was continued. After trypsin digestion, the samples were desalted
with TARGA C18 columns (Nest Group, Southborough, MA) and resuspended
in 5% acetonitrile (ACN) and 5% formic acid (FA) prior to analysis.
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