Purified viruses were treated using the PNGaseF kit (NEB) according to the manufacturer’s instructions. The samples were then treated with 4X-Laemli buffer (Bio-Rad) containing beta-mercaptoethanol (BME) and heated at 100 °C for 5 min. Approximately 1 ug purified virus was added to each lane on a 7.5% Mini-PROTEAN® TGX™ precast protein gel (Bio-Rad) and run for 5.5 h at 25 V. After electrophoresis, the proteins were transferred to a nitrocellulose membrane using the iBlot transfer and stack device (Invitrogen) at 20 V for 7 min. The membranes were blocked in 5% nonfat dry milk (Bio-Rad) in PBS-T (0.5%) for 1 h at room temperature with shaking. The blocking solution was removed and anti-N2 guinea pig antisera (generated in-house by vaccinating guinea pigs intramuscularly with Hong Kong14 N2 recombinant protein) diluted 1:2000 in PBS supplemented with 1% (w/v) BSA was added. The membranes were incubated overnight and washed three times with PBS-T. The following day, a donkey anti-guinea pig IgG-horseradish peroxidase conjugate (IgG-HRP; EMD Millipore, AP193P) was added for 1 h at room temperature and the membranes were washed. The membranes were developed by adding ECL prime and incubated for 5 min. The developed blot membranes were visualized by scanning using an ImageScanner III imager with accompanying LabScan software (GE Healthcare).
Pngase f kit
Manufactured by New England Biolabs
Sourced in United States
PNGase F is an enzyme that cleaves the bond between asparagine and the N-acetylglucosamine residue of N-linked glycoproteins. The PNGase F kit provides the necessary components for the deglycosylation of N-linked glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
Pngase f, by New England Biolabs (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Labscan, by GE Healthcare (1 mentions)
Laemli buffer, by Bio-Rad (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Abts solution, by Roche (1 mentions)
Criterion any kd tgx gel, by Bio-Rad (1 mentions)
Bcip nbt, by Merck Group (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Dowex column, by Merck Group (1 mentions)
Ripa lysis buffer, by Nacalai Tesque (1 mentions)
Supersignal west femto system, by Thermo Fisher Scientific (1 mentions)
Las 4000 mini camera system, by Fujifilm (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Celltiter glo luminescent, by Promega (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
Bio glo luciferase assay reagent, by Promega (1 mentions)
2 aminobenzamide 2 ab, by Merck Group (1 mentions)
17 protocols using pngase f kit
1
SDS-PAGE and Western Blot Analysis of Viral Proteins
2
Deglycosylation and ELISA Analysis
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All ELISAs were performed on Costar high bind 96-well plates. For ELISA, coating of single chain recombinant proteins (0.5 μg/well) was followed by washing, blocking with PBS/1%BSA/0.05% tween 20 (Merck), incubation with detecting antibody, washing, incubation with polyclonal rabbit anti mouse IgG-HRP (Roche), washes with PBS/0.05% tween and PBS, and incubation with ABTS solution (Roche). ELISA plates were read at 405 nm. Bovine single chain CD1b3 was deglycosylated using the PNGaseF kit from New England Biolabs. The native deglycosylation reaction was performed in G7 reaction buffer for five days at 37°C for 5 days. For mock treated protein, PNGase was omitted, but otherwise treated equally. The same samples that were used for ELISA were analyzed on a denaturing 14% SDS-page gel followed by Coomassie blue staining, together with samples that were deglycosylated in glycoprotein denaturing buffer, NP40 buffer, and G7 reaction as a control for maximum deglycosylation obtained under denaturing conditions.
3
Enzymatic Deglycosylation of Proteins
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Deglycosylation reaction was performed following instructions from the PNGase F kit purchased from New England Biolabs, Ipswich, MA (P0705). Briefly, 20 μg of total protein from tissue supernatant was first denatured in 1× denaturation buffer at 100 °C for 10 min. Then the denatured protein was deglycosylated with 500 units of PNGsae F in 1× Glyco-buffer and 1% NP-40 at 37 °C for an hour. Negative control reaction without the addition of PNGase F was prepared for each sample for side-by-side comparison of glycosylation status. The reaction was quenched by SDS sample buffer and reducing reagent mix, then resolved by SDS-PAGE with 10–20% Tris-glycine gels for subsequent LAMP1 detection by Western blotting mentioned previously.
4
Deglycosylation of IFNλ4 Variants
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N-glycans of IFNλ4 were removed using a PNGase F kit (#P0704S, New England Biolabs) according to the manufacturer’s instructions. Briefly, IFNλ4 variants were boiled with Glycoprotein Denaturing Buffer (10×) and chilled on ice. GlycoBuffer(10×), NP-40(10×), and 1 μl of PNGase F were added onto denatured proteins and the mixture was incubated at 37 °C for 1 h before the Western blot analysis.
5
Protein Deglycosylation and SDS-PAGE Analysis
6
PfRH5 Glycosylation Analysis
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The presence of glycosylation on the PfRH5 variant proteins was determined using the PNGase F kit (New England Biolabs, Herts, UK) under denaturing reaction conditions following the supplied protocol. Briefly, 2 μg protein was first heated to 95 °C for 5 min in the Glycoprotein Denaturing Buffer containing 0.5% SDS and 40 mM DTT, then chilled on ice before the addition of 1% NP-40, GlycoBuffer 2, and PNGase F, and then incubated at 37 °C for 1 h. Reaction products were separated on a Criterion Any kD TGX gel (Bio-Rad Laboratories, Herts, UK) using Tris-Glycine-SDS running buffer as previously described, and subsequently transferred to nitrocellulose membrane using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Herts, UK). Detection of PfRH5 proteins was performed using full-length PfRH5-reactive rabbit serum7 (link) as the primary antibody, and alkaline-phosphatase labelled donkey anti-rabbit (Jackson ImmunoResearch, Suffolk, UK) as the secondary antibody. Blots were developed using BCIP/NBT (Sigma-Aldrich, Dorset, UK). The Western blot of the v1.0 PfRH5 stable cell line supernatant in Fig. S1B used the same methodology but with reducing and non-reducing conditions.
7
Glycan Extraction and Purification Protocol
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Cell pellets were resuspended in ammonium bicarbonate and passed several times through a 23-gauge syringe to homogenize. Cells were further lysed with probe sonication and the lysate was denatured by adding 10 μL of denaturing buffer (NEB PNGase F kit, P0709) and heating at 100 °C for 5 min. Salts were removed with a pre-washed 10 kDa MWCO filter and exchanged into 50 mM ammonium bicarbonate. Whole proteins were removed from the top of the MWCO filter and resuspended in 50 mM ammonium bicarbonate. N-glycans were released with PNGase F (NEB, P0709) for 20 h at 37 °C and the released N-glycans were isolated by passage through a pre-washed 10 kDa MWCO filter. The remaining protein including O-linked glycans remained in the top of the filter and were removed before treated with 1 M sodium borohydride in 50 mM sodium hydroxide. O-linked glycans were released with beta-elimination at 45 °C overnight. The basic solution was neutralized with a 10 % acetic acid solution, desalted through a packed Dowex column (50 W x8-100, Sigma Aldrich, St. Louis, MO) then lyophilized. Borates were removed from the sample with repeated additions of 9:1 methanol: acetic acid under a nitrogen flow. The material was then resolubilized and pass through a C18 SPE cartridge to further purify the material before permethylation and analysis.
8
Detecting Syncytin-2 Expression in Transfected Cells
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Syncytin‐2 expression plasmids were transfected into 293T and G355‐5 cells in the same manner as cell fusion assays. Cells were lysed in RIPA Lysis Buffer (#08714‐04; Nacalai Tesque) from transfection to G355‐5 cells at 7 h and from transfection to 293T cells at 20 h. Cellular suspensions were subjected to glycolysis treatment for 1 h using a PNGase F Kit (New England BioLabs). SDS/PAGE was performed using Mini‐PROTEAN TGX Precast Gels (#4561094; Bio‐Rad Laboratories, Inc., Hercules, CA, USA). Peptides from the gel were transferred to polyvinylidene difluoride membranes, and the monoclonal ANTI‐FLAG M2 antibody (#F3165; Sigma‐Aldrich) was used to detect Flag‐tagged syncytin‐2. Signals were detected using a Super Signal West Femto System (#34095; Thermo Fisher Scientific), and images were obtained using a LAS4000 Mini camera system (Fujifilm, Tokyo, Japan).
9
Characterization of Perjeta® Antibody Batches
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A total of 13 batches of Perjeta® (420 mg/vial) were purchased from China, and the corresponding batch numbers and expiration date are shown in Table 1 .
The CellTiter-Glo Luminescent and Bio-Glo Luciferase Assay Reagent was obtained from Promega (Madison, WI, USA). Goat anti-human IgG- (Fc specific) peroxidase antibody, sodium dodecyl sulfate (SDS), n-ethyl maleimide (NEM), sodium cyanoborohydride, 2-aminobenzamide (2-AB), and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HER2 antigen was purchased from Sino Biological Co., Ltd. (Beijing, China). The PNGase F kit was obtained from New England Biolabs (Ipswich, MA, USA). The Measure-iT™ Thiol Assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). FabALACTICA (IgdE) was obtained from Genovis (Lund, Sweden). TSKgel FcR-IIIA-NPR column was obtained from Tosoh Bioscience (Tokyo, Japan).
The CellTiter-Glo Luminescent and Bio-Glo Luciferase Assay Reagent was obtained from Promega (Madison, WI, USA). Goat anti-human IgG- (Fc specific) peroxidase antibody, sodium dodecyl sulfate (SDS), n-ethyl maleimide (NEM), sodium cyanoborohydride, 2-aminobenzamide (2-AB), and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HER2 antigen was purchased from Sino Biological Co., Ltd. (Beijing, China). The PNGase F kit was obtained from New England Biolabs (Ipswich, MA, USA). The Measure-iT™ Thiol Assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). FabALACTICA (IgdE) was obtained from Genovis (Lund, Sweden). TSKgel FcR-IIIA-NPR column was obtained from Tosoh Bioscience (Tokyo, Japan).
10
Characterization of Biosimilar SB3 Compared to Reference Products
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EU- and US-sourced reference product (up to 154 lots) were purchased from local distributors and then stored according to the manufacturer’s instructions. The reference standard for the bioassays was prepared from EU-sourced reference product and used for characterization of the reference products and SB3. The CellTiter-Blue® and CytoTox-Glo® kits were obtained from Promega (Madison, WI, USA). The PNGase-F kit was obtained from New England Biolabs (NEB, Ipswich, MA, USA). The 2-aminobenzamide (2-AB) labelling kit for N-glycan analysis was obtained from Ludger (Oxfordshire, UK). Glutathione (GSH)-coated donor beads, human IgG-conjugated acceptor beads, and streptavidin-coated donor beads were obtained from PerkinElmer (Waltham, MA, USA). The glutathione-S-transferase (GST)-tagged FcγRIIIa and HER2-Fc were obtained from Biogen (Cambridge, MA, USA). Biotin-labelled neonatal Fc receptor (FcRn) was obtained from Biogen (Cambridge, MA, USA). C1q protein was obtained from Quidel (San Diego, CA, USA). Anti-C1q horseradish peroxidase (HRP) conjugated antibody (catalog number AB46191) was obtained from Abcam (Cambridge, MA, USA). Statistical analysis and graphical comparison were performed with Minitab statistic software package (Leadtools Technologies Inc., version 18.1.0, Charlotte, NC, USA).
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