Three to five mg of purified T. reesei EG1 expressed in S. cerevisiae was incubated in pH 7.0, 25 mM phosphate buffer along with 200 units of PNGase F (NEB) at 30°C for 18 h following which an additional 100 units of PNGase F were added and incubated for another 24 h. PNGase F treated T. reesei EG1 was purified using gel filtration chromatography.
Pngase f
Manufactured by New England Biolabs
Sourced in United States, United Kingdom, Germany, Japan, Canada, France, China, Morocco, Italy
PNGase F is an enzyme that cleaves the bond between the asparagine residue and the N-acetylglucosamine residue in N-linked glycoproteins. It is commonly used in the analysis and characterization of glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
Endo h, by New England Biolabs (183 mentions)
O glycosidase, by New England Biolabs (44 mentions)
Glycoprotein denaturing buffer, by New England Biolabs (31 mentions)
Neuraminidase, by New England Biolabs (27 mentions)
Trypsin, by Promega (23 mentions)
Glycobuffer 2, by New England Biolabs (21 mentions)
Endo hf, by New England Biolabs (21 mentions)
Formic acid, by Merck Group (19 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (19 mentions)
Iodoacetamide, by Merck Group (18 mentions)
Dithiothreitol (dtt), by Merck Group (16 mentions)
Protease inhibitor cocktail, by Roche (13 mentions)
Sequencing grade trypsin, by Promega (13 mentions)
Ammonium bicarbonate, by Merck Group (12 mentions)
Protease inhibitor cocktail, by Merck Group (12 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Endoglycosidase h, by New England Biolabs (11 mentions)
Acetonitrile, by Merck Group (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Trypsin, by Merck Group (10 mentions)
1 106 protocols using pngase f
1
Deglycosylation of Recombinant EG1
2
Enzymatic Release and Analysis of N-Glycans from HIV-1 Envelope Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Enzymatic release of N-linked glycans from HIV-1 gp145 was carried out following the method of Küster et al. [31] (link) with minor modifications. Briefly, excised bands containing 20 µg of gp145 were incubated with 5000 units of PNGase F (NEB) for 24 hrs at 37°C [31] (link). For the analysis of oligosaccharides containing sialic acid, 20 µg of gp145 in solution were incubated with 50 units of α2-3,6,8,9 NEUA (NEB) for 24 hrs at 37°C, prior to PNGase F digestion. Subsequently, glycans were extracted into LC-MS water in an ultrasonic bath. The extracted glycans were then dried in a vacuum concentrator and dissolved with water. For MALDI-ToF analysis, glycans were co-crystallized with a solution of 50% acetonitrile/50% 20 mM ammonium citrate containing 134 mM of 2′,4′,6′-Trihydroxyacetophenone monohydrate (Sigma-Aldrich). MALDI-ToF MS analysis of the N-glycans was performed in the reflector positive and negative ion mode using a 4800 Plus MALDI ToF/ToF Analyzer (AB Sciex) that was calibrated externally using the Calmix 5 Opti-ToF High-Resolution TIS Calibration Insert (AB Sciex). N-glycan assignments were carried out using GlycoWorkbench software [32] (link).
3
Glycosidase Digestion Analysis of Immunoprecipitated Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Endo H digestion, eluted fractions of the immunoprecipitants from the cell lysate or culture sup were first diluted with 1× glycoprotein denaturing buffer (0.5% SDS and 40 mM dithiothreitol [DTT]) and boiled at 98 °C for 10 min. The denatured samples were divided into two halves. Half of the sample was diluted with 1× GlycoBuffer 3 (50 mM sodium acetate pH 6.0) (New England Biolabs) and then treated with Endo H (New England Biolabs) at 37 °C for 4.5 h. For PNGase F digestion, the other half of the samples were diluted with 1× GlycoBuffer 2 (50 mM sodium phosphate pH 6.0) and 1% NP-40 and then treated with PNGase F (New England Biolabs) at 37 °C for 4.5 h. Enzyme-treated samples were separated by 12% SDS-PAGE under reducing conditions and analyzed by Western blotting using the anti-FLAG tag mAb.
4
Deglycosylation of Membrane Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PNGase F and O-glycosidase were purchased from New England BioLabs and used according to the manufacturer’s instructions. Flp-In TREx293 (tet-on) cells or mouse hypothalamus cell membranes were incubated with a mixture of cholesteryl hemisuccinate (1%) and dodecyl-β-d-maltoside (0.2%), and their soluble fractions were denatured using 1 × Glycoprotein Denaturing Buffer (New England BioLabs) for 5 min at 70 °C. After chilled on ice, the denatured proteins were incubated with PNGase F (50 U/µl) or O-glycosidase (400 U/µl) in 1 × G7 Reaction Buffer (New England BioLabs) supplemented with 1% NP-40 for 2 h at 37 °C. The reaction was stopped by Laemmli buffer for the subsequent analysis.
5
Enzymatic Deglycosylation of Glycopeptides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
N-linked glycans were digested by Endo F3 (Millipore Sigma) followed by PNGase F (New England BioLabs). First, label-free or TMT-labeled glycopeptides were resuspended in 100 mM ammonium acetate (pH 5.5) and treated with Endo F3 at an enzyme/peptides ratio of 25 milliunits/80 μg of glycopeptides (50 milliunits/80 μg of glycopeptides for HCC tumor and normal tissues). The enzymatic reaction was conducted at 37 °C for 2 hours. The samples were dried in a speed vacuum centrifuge for 5 hours. The dried peptides were redissolved in 100 mM ammonium bicarbonate (pH 8) and treated with PNGase F (New England BioLabs) at an enzyme/peptides ratio of 3000 New England Biolabs units/80 μg of glycopeptides. The reaction solution was incubated at 37 °C for 2 hours. The resulting peptides were desalted with C18 solid-phase extraction.
6
Immunoprecipitation and Deglycosylation of FPN-V5
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysates from doxycycline-induced T-REx™/FPN-V5 cells (370 μg of protein) were immunoprecipitated with anti-V5 antibody (Abcam) and protein G Dynabeads (Thermo Fisher). Immunoprecipitates were treated with PNGase F (New England BioLabs) according to New England BioLabs protocol with the following modifications: (1) denaturation at 50°C for 30 min and (2) PNGase F treatment for 3 h at 37°C. Samples were electrophoresed on a 4–20% Tris-Glycine gel, transferred to nitrocellulose and probed with Abs 38C8 (20 μg/ml) and 38G6 (1 μg/ml).
7
In vitro Deglycosylation of Glycoproteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro deglycosylation with Endo H (P0702S, New England Biolabs) or PNGase F (P0704S, New England Biolabs) was carried out as described previously [34 (link)]. Briefly, 20 μg glycoprotein was heated at 100 °C for 10 min, combined with 1 μL of Glycoprotein Denaturing Buffer (B1704, New England Biolabs). The denatured samples were incubated at 37 °C for 1 h with 1000 units of Endo H or 500 units of PNGase F in a total reaction volume of 20 μL, containing 2 μL glycoBuffer 3 (B1720, New England Biolabs), or 2 μL glycoBuffer 2 (B3704, New England Biolabs) and NP40 (B2704, New England Biolabs) respectively. Samples were analyzed by western blotting.
8
Glycan Removal Optimization for Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Removal of N-linked glycans was performed using PNGaseF (New England
Biolabs, Ipswich, USA) at a concentration of 125 U/μg protein.
Removal of O-linked glycans was performed using O-glycosidase (New
England Biolabs, 20,000 U/μg protein), α2-3,6,8 neuraminidase
(New England Biolabs, 25 U/μg protein), and α-N-acetyl-galactosaminidase (New England Biolabs, 10 U/μg
protein). Removal of both N-linked and O-linked glycans was performed
using PNGaseF (New England Biolabs, 125 U/μg protein), O-glycosidase
(New England Biolabs, 20,000 U/μg protein), α2-3,6,8 neuraminidase
(New England Biolabs, 25 U/μg protein), and α-N-acetyl-galactosaminidase (New England Biolabs, 10 U/μg
protein). Removal of sialic acids was performed using the α2-3,6,8
neuraminidase (New England Biolabs, 50 U/μg protein). Removal
of fucose was performed using α1-2,4,5,6 fucosidase O (New England
Biolabs, 2 U/μg protein) and α1-3,4 fucosidase (New England
Biolabs, 4 U/μg protein). All enzymatic reactions were performed
as a 1-step reaction with 1× Glycobuffer 2 (New England Biolabs)
and 10 μg of RBD produced in CHO-S-, HEK293F-, or Lec3.2.8.1
cells and incubation at 37 °C for 24 h. As heat-treated controls,
peptides were incubated at 37 °C for 24 h but without additional
enzymes.
Biolabs, Ipswich, USA) at a concentration of 125 U/μg protein.
Removal of O-linked glycans was performed using O-glycosidase (New
England Biolabs, 20,000 U/μg protein), α2-3,6,8 neuraminidase
(New England Biolabs, 25 U/μg protein), and α-N-acetyl-galactosaminidase (New England Biolabs, 10 U/μg
protein). Removal of both N-linked and O-linked glycans was performed
using PNGaseF (New England Biolabs, 125 U/μg protein), O-glycosidase
(New England Biolabs, 20,000 U/μg protein), α2-3,6,8 neuraminidase
(New England Biolabs, 25 U/μg protein), and α-N-acetyl-galactosaminidase (New England Biolabs, 10 U/μg
protein). Removal of sialic acids was performed using the α2-3,6,8
neuraminidase (New England Biolabs, 50 U/μg protein). Removal
of fucose was performed using α1-2,4,5,6 fucosidase O (New England
Biolabs, 2 U/μg protein) and α1-3,4 fucosidase (New England
Biolabs, 4 U/μg protein). All enzymatic reactions were performed
as a 1-step reaction with 1× Glycobuffer 2 (New England Biolabs)
and 10 μg of RBD produced in CHO-S-, HEK293F-, or Lec3.2.8.1
cells and incubation at 37 °C for 24 h. As heat-treated controls,
peptides were incubated at 37 °C for 24 h but without additional
enzymes.
9
Enzymatic Deglycosylation of GABA Uptake
10
Glycoprotein Analysis by Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified LAMP-1 were run on SDS-PAGE and stained with Coomassie blue R250. The corresponding protein bands were then excised from the gel and degylcosylated by in-gel PNGase F treatment. For in-gel PNGase F digestion, the gel slices were first reduced with 10 mM DTT at 60 °C for 1 h and alkylated with 55 mM iodoacetamide at RT for 1 h followed by soaking with one unit of PNGase F (P0705L, New England Biolabs, Ipswich, MA, USA) in 25 mM ammonium bicarbonate buffer at 37 °C overnight. Afterward, N-glycans were extracted two times with pure water and 50% acetonitrile per each. All extracts were pooled together and concentrated by a SpeedVac. The remaining gels were further digested with 12.5 ng/μl trypsin (V5111 Promega, Madison, WI, USA) in 25 mM NH4HCO3 buffer and incubated overnight at 37 °C. The peptide fragments extracted from gels were used for peptide mapping.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!