The surface N-glycans of iWJ-MSC-EV were removed by incubation with the enzyme PNGase-F. Specifically, 10 U of PNGase-F (N-glycosidase F; Roche Diagnostics GmbH, Mannheim, Germany) were added to 100 µL of CCM and incubated for 18 h at 37 °C before EV isolation. The enzyme was eliminated by the downstream SEC purification of iWJ-MSC-EV.
Pngase f
Manufactured by Roche
Sourced in Germany, United States, Switzerland, Australia
PNGase F is an enzyme that catalyzes the cleavage of asparagine-linked glycosidic linkages in glycoproteins and glycopeptides. It is commonly used in the analysis and characterization of glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Roche (5 mentions)
Sialidase, by Roche (5 mentions)
Formic acid, by Merck Group (5 mentions)
Dithiothreitol (dtt), by Merck Group (5 mentions)
Asp n, by Roche (4 mentions)
Rapigest, by Waters Corporation (4 mentions)
Endoh, by Roche (4 mentions)
Mwco centrifugal filter columns, by Merck Group (3 mentions)
Glu c, by Roche (3 mentions)
Iodoacetamide, by Merck Group (3 mentions)
Sequencing grade trypsin, by Promega (3 mentions)
Sep pak c18, by Waters Corporation (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Acetic acid, by Merck Group (3 mentions)
Rprotein a ff, by Cytiva (2 mentions)
Endo h, by New England Biolabs (2 mentions)
Blotglyco, by Sumitomo Bakelite (2 mentions)
Vivaspin 500, by Sartorius (2 mentions)
Anti tetra his mab, by Qiagen (2 mentions)
Sequencing grade modified trypsin, by Promega (2 mentions)
109 protocols using pngase f
1
N-glycan Removal From iWJ-MSC-EV
2
Enzymatic Deglycosylation of Cell Lysates
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For PNGase F (Roche) treatment, 1 μL of 1000 U/mL PNGase F was added to 17 μL of cell lysate with 2 μL of 0.5 M Tris-HCl (pH 8.0). For Endo H (Roche) treatment, 1 μL of 5 U/mL Endo H was added to 17 μL of cell lysate with 100 mM sodium citrate (pH 5.5). The mixtures were incubated at 37°C for 1 h. The samples were then denatured in sample buffer at 37°C for 30 min and analysed by western blotting.
3
Purification and Deglycosylation of IgG Antibodies
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Example 40
Purification of IgG, IgG4 and IgG4-Hingeless Antibodies
All IgG1, IgG4 and hingeless antibodies were purified. First the supernatants were filtered over 0.20 μM dead-end filter. Then, the supernant was loaded on a 5 ml Protein A column (rProtein A FF, Amersham Bioscience) and eluted with 0.1 M citric acid-NaOH, pH 3. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9 and dialyzed overnight to 12.6 mM sodium phosphate, 140 mM NaCl, pH 7.4 (B. Braun, Oss, The Netherlands). After dialysis samples were sterile filtered over 0.20 μM dead-end filter.
Antibodies were deglycosylated by overnight incubation at 37° C. with 1 unit PNgase F (Roche)/μg antibody, followed by purification on protein A.
Samples were analysed for concentration of IgG by nephelometry and absorbance at 280 nm.
4
Galectin-1 Binding to VEGFR-2 and NRP-1
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Recombinant galectin-1 was labeled with biotin using an EasyLink Biotin Conjugation Kit (ab102865; Abcam), according to the manufacturer’s instructions. Proteins immunoprecipitated with anti-VEGFR-2 or NRP-1 antibody as described above were used for the galectin-1 overlay assay. Some immunoprecipitated VEGFR-2 proteins from the lysates for the mid CL were digested with 25 U/ml of PNGase F (1365169; Roche) according to the manufacturer’s instructions to release asparagine-linked glycans (N-glycans) from glycoproteins.
Immunoprecipitated VEGFR-2 proteins with or without PNGase F treatment were separated by SDS-PAGE and subsequently transferred to a PVDF membrane. The early CL was used as a negative control. The membrane was blocked with 5% BSA in TBS-T and then exposed to biotin-conjugated recombinant galectin-1 diluted at 1:300 for 2 h at room temperature. After washing with TBS-T, the membrane was incubated with HRP-conjugated streptavidin, and the reactive protein bands were detected as described above.
Immunoprecipitated VEGFR-2 proteins with or without PNGase F treatment were separated by SDS-PAGE and subsequently transferred to a PVDF membrane. The early CL was used as a negative control. The membrane was blocked with 5% BSA in TBS-T and then exposed to biotin-conjugated recombinant galectin-1 diluted at 1:300 for 2 h at room temperature. After washing with TBS-T, the membrane was incubated with HRP-conjugated streptavidin, and the reactive protein bands were detected as described above.
5
Glycopeptide Deglycosylation and Desalting
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The dried 20 μg glycopeptides were redissolved in 50 mM Tris–HCl pH 8.0, and 2 units of peptide-N-glycosidase F (PNGaseF; Roche Inc., Basel, Switzerland) were added and incubated at 37 ℃ for 16 h. Desalting was performed with a C-tip SDB (Nikkyo Technos Int., Tokyo, Japan). The deglycosylated peptides were dried with the Speed Vac concentrator (Sakuma). Lastly, the dried glycopeptides were resuspended in 3% acetonitrile/0.1% formic acid.
6
Protein Mass Analysis Protocol
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The molecular masses of intact samples were measured using a quadrupole time-of-flight mass spectrometer (Q-TOF MS; Synapt G2, Waters, USA) coupled with an Acquity ultra-performance liquid chromatography (UPLC) instrument (Waters). The spectra data were deconvoluted by the BiopharmaLynx™ software (Waters). For molecular mass analysis of reduced and deglycosylated samples, the samples were pretreated with PNGase F (Roche, Germany) to remove N-linked glycans, and the disulfides were reduced with dithiothreitol.
7
Glycan Deglycosylation and Sialic Acid Labeling
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Two microliters of human serum were denatured for 10 min at 50°C in the presence of four microliters of 1% SDS (w/w) (Merck Millipore, Germany). Thereafter, four microliters of a releasing solution containing 2% NP-40 (Calbiochem, CA, USA) (w/w) and 0.5 mU PNGase F (EC 3.5.152; Roche Applied Science, Indianapolis, IN) in 2.5 × PBS (10xPBS containing 57 g/L Na2HPO4 × 2H2O, 5 g/L KH2PO4, and 85 g/L NaCl, pH 7.4) were added to the samples and incubated for 5 h at 37°C. Samples were then stored at −20°C until sialic acids were labeled.
8
Deglycosylation of IgG1 for Mass Spectrometry
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Example 7
As there is heterogeneity in the glycosylation of the IgGs, the proteins were deglycosylated in order to create a single product with a distinct mass, suitable for mass spectrometric analysis. One unit of N-glycosidase F (PNGase F; Roche Diagnostics, Mannheim, Germany) was incubated per 10 μg of IgG1, overnight at 37° C. Buffer exchange using 10 kDa MWCO centrifugal filter columns (Millipore) was performed to remove the original purification buffer (0.1 M citrate buffer pH 3.0/1.0 M Tris-HCL pH 8.0) and to rebuffer to PBS. Similar buffer exchange procedures were performed to remove the detached glycan chains, and to change the buffer to 150 mM ammonium acetate pH 7.5. Filters were washed with 200 μl 150 mM ammonium acetate pH 7.5, for 12 min 11,000 rpm and 4° C. After washing 50 μl deglycosylated IgG was loaded on the filter and 450 μl of 150 mM ammonium acetate pH 7.5 was added, subsequently followed by another centrifugation round of 12 min at 11,000 rpm at 4° C. In total the centrifugation was repeated 5 times, each time fresh 150 mM ammonium acetate pH 7.5 buffer was added to a total volume of 500 μl. After the last centrifugation step the remaining buffer exchanged deglycosylated IgG1, approximately 25 μl, was collected and transferred to an eppendorf tube, ready for mass spectrometric analysis.
9
Validating N-Glycosylation of Panx2 Protein
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Lysates from HEK293T cells ectopically expressing mouse Panx2 and the N86Q were used for validation of N-glycosylation site of the mouse Panx2 construct. Enzymatic de-glycosylation with Peptide-N-glycosidase F (PNGase F) and Endoglycosidase H (EndoH) were used to detect the presence of all complex forms of N-glycosylation and high-mannose modification, respectively. PNGase F (Roche, Indianapolis, IN, USA) and EndoH (New England Biolabs Ipswich, MA, USA) digestions were performed according to their manufacturer’s instructions. Briefly, at least 35 µg of total protein was denatured at 100 °C for 5 min in denaturing buffer (0.1% (v/v) SDS, 0.05 M 2-mercaptoethanol, 50 mM phosphate buffer, pH 7.5) and subsequently incubated for 1h at 37 °C with 10 units of the PNGase F, 0.7% (v/v) of Triton X-100 or 2500 U of Endo H in supplier’s digestion buffer. In the parallel control, samples were assayed without endoglycosidases. Protein samples were separated on an 8% SDS-polyacrylamide gel electrophoresis gel (PAGE) and transferred to nitrocellulose membranes for WB.
10
Profiling PDAC Cell Line O-Glycans
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O-Glycans present in PDAC cell lines were analysed as described before28 (link). Briefly, lysed cell pellets containing 500,000 cells were loaded to preconditioned PVDF membrane plate (Millipore) wells and denatured with guanidine hydrochloride and dithiothreitol (DTT). After removing the denaturation agent, N-glycans were released by PNGase F (Roche) digestion overnight at 37 °C. Upon removal of N-glycans, 50 μL of 0.5 M sodium borohydride in 50 mM potassium hydroxide was added to each well and incubated for 16 h at 50 °C for the release of O-glycans via reductive beta-elimination. Upon recovery of the released O-glycans, samples were desalted by cation exchange solid-phase extraction (Dowex (50W-X8), Sigma- Aldrich). Desalted O-glycans were further purified via solid-phase extraction by packing bulk sorbent PGC slurry (Grace Discovery sciences) into 96-well filter plates. Analysis was performed using a PGC nano-LC–ESI-MS/MS platform. Identification of glycans was performed based on PGC retention time, known biosynthetic pathways, and manual inspection of MS/MS spectra following known fragmentation pathways of O-glycan alditols in negative-ion mode. Relative quantitation was performed on the total area of all O-glycans within one sample normalising it to 100%.
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