endohf  (New England Biolabs)


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  • 94
    Name:
    Endo Hf
    Description:
    Endo Hf 500 000 units
    Catalog Number:
    p0703l
    Price:
    296
    Category:
    Glycosidases
    Size:
    500 000 units
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    Structured Review

    New England Biolabs endohf
    Endo Hf
    Endo Hf 500 000 units
    https://www.bioz.com/result/endohf/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    endohf - by Bioz Stars, 2021-03
    94/100 stars

    Images

    1) Product Images from "The intellectual disability protein RAB39B selectively regulates GluA2 trafficking to determine synaptic AMPAR composition"

    Article Title: The intellectual disability protein RAB39B selectively regulates GluA2 trafficking to determine synaptic AMPAR composition

    Journal: Nature Communications

    doi: 10.1038/ncomms7504

    PICK1, AMPARs neuronal localization, maturation and GluA2 surface expression. ( a ) Quantification of PICK1 (shScramble n =21 cells; shRab39b n =21 cells; Rab39b-rescue n =8 cells; Student’s t -test shScramble versus shRab39b P =0.009; Rab39b-rescue versus shRab39b P =0.002), GluA1 (shScramble n =10 cells; shRab39b n =10 cells; Rab39b-rescue n =9 cells), GluA2 (shScramble n =9 cells; shRab39b n =5 cells; Rab39b-rescue n =11 cells; Student’s t -test shScramble versus shRab39b P =0.003; Rab39b-rescue versus shRab39b P =0.004) and GluA3 (shScramble n =6 cells; shRab39b n =7 cells; Rab39b-rescue n =9 cells; Student’s t -test shScramble versus shRab39b P =0.03; Rab39b-rescue versus shRab39b P =0.04) cell body density in shScramble-, shRab39b- and Rab39b-rescue-treated mouse hippocampal neurons. ( b ) Quantification of the ratio between mature (1) and immature (2) forms of AMPARs in shRab39b- compared with shScramble-treated neurons after PNGasef or EndoHf digestion. NT: non-treated neurons. Representative western blots (lower panels) showing the maturation ratio for GluA1 ( n =3 experimental replicates), GluA2 ( n =3 experimental replicates; Student’s t -test P =0.002) and GluA3 ( n =3 experimental replicates; Student’s t -test P =1.28E−04). ( c ) Quantification of PICK1 (shScramble n =14 cells; shRab39b n =15 cells; Rab39b-rescue n =8 cells; Student’s t -test shScramble versus shRab39b P =0.03; Rab39b-rescue versus shRab39b P =0.01), GluA1 (shScramble n =10 cells; shRab39b n =10 cells; Rab39b-rescue n =9 cells; Student’s t -test shScramble versus shRab39b P =7.4E−04; Rab39b-rescue versus shRab39b P =8.1E−04), GluA2 (shScramble n =10 cells; shRab39b n =5 cells; Rab39b-rescue n =9 cells; Student’s t -test shScramble versus shRab39b P =2.8E−07; Rab39b-rescue versus shRab39b P =3.1E−05) and GluA3 (shScramble n =6 cells; shRab39b n =7 cells; Rab39b-rescue n =9 cells; Student’s t -test shScramble versus shRab39b P =1.5E−05; Rab39b-rescue versus shRab39b P =4.7E−05) dendrite density in shScramble-, shRab39b- and Rab39b-rescue-treated mouse hippocampal neurons. ( d ) Representative images of shRab39b, shScramble and Rab39b-rescue neurons immunostained without permeabilization for the extracellular N-terminal region of GluA1 and GluA2. Quantification of positive puncta at cell surface shows a significant increase of GluA1 (shScramble n =41 cells; shRab39b n =40 cells; Rab39b-rescue n =10; Student’s t -test shScramble versus shRab39b P =0.03; Rab39b-rescue versus shRab39b P =0.006) and significant decrease of GluA2 (shScramble n =89 cells; shRab39b n =70 cells; Rab39b-rescue n =8; Student’s t -test shScramble versus shRab39b P =0.006; Rab39b-rescue versus shRab39b P =0.01) subunits. The number of cells belongs from a minimum of three experimental replicates. * P
    Figure Legend Snippet: PICK1, AMPARs neuronal localization, maturation and GluA2 surface expression. ( a ) Quantification of PICK1 (shScramble n =21 cells; shRab39b n =21 cells; Rab39b-rescue n =8 cells; Student’s t -test shScramble versus shRab39b P =0.009; Rab39b-rescue versus shRab39b P =0.002), GluA1 (shScramble n =10 cells; shRab39b n =10 cells; Rab39b-rescue n =9 cells), GluA2 (shScramble n =9 cells; shRab39b n =5 cells; Rab39b-rescue n =11 cells; Student’s t -test shScramble versus shRab39b P =0.003; Rab39b-rescue versus shRab39b P =0.004) and GluA3 (shScramble n =6 cells; shRab39b n =7 cells; Rab39b-rescue n =9 cells; Student’s t -test shScramble versus shRab39b P =0.03; Rab39b-rescue versus shRab39b P =0.04) cell body density in shScramble-, shRab39b- and Rab39b-rescue-treated mouse hippocampal neurons. ( b ) Quantification of the ratio between mature (1) and immature (2) forms of AMPARs in shRab39b- compared with shScramble-treated neurons after PNGasef or EndoHf digestion. NT: non-treated neurons. Representative western blots (lower panels) showing the maturation ratio for GluA1 ( n =3 experimental replicates), GluA2 ( n =3 experimental replicates; Student’s t -test P =0.002) and GluA3 ( n =3 experimental replicates; Student’s t -test P =1.28E−04). ( c ) Quantification of PICK1 (shScramble n =14 cells; shRab39b n =15 cells; Rab39b-rescue n =8 cells; Student’s t -test shScramble versus shRab39b P =0.03; Rab39b-rescue versus shRab39b P =0.01), GluA1 (shScramble n =10 cells; shRab39b n =10 cells; Rab39b-rescue n =9 cells; Student’s t -test shScramble versus shRab39b P =7.4E−04; Rab39b-rescue versus shRab39b P =8.1E−04), GluA2 (shScramble n =10 cells; shRab39b n =5 cells; Rab39b-rescue n =9 cells; Student’s t -test shScramble versus shRab39b P =2.8E−07; Rab39b-rescue versus shRab39b P =3.1E−05) and GluA3 (shScramble n =6 cells; shRab39b n =7 cells; Rab39b-rescue n =9 cells; Student’s t -test shScramble versus shRab39b P =1.5E−05; Rab39b-rescue versus shRab39b P =4.7E−05) dendrite density in shScramble-, shRab39b- and Rab39b-rescue-treated mouse hippocampal neurons. ( d ) Representative images of shRab39b, shScramble and Rab39b-rescue neurons immunostained without permeabilization for the extracellular N-terminal region of GluA1 and GluA2. Quantification of positive puncta at cell surface shows a significant increase of GluA1 (shScramble n =41 cells; shRab39b n =40 cells; Rab39b-rescue n =10; Student’s t -test shScramble versus shRab39b P =0.03; Rab39b-rescue versus shRab39b P =0.006) and significant decrease of GluA2 (shScramble n =89 cells; shRab39b n =70 cells; Rab39b-rescue n =8; Student’s t -test shScramble versus shRab39b P =0.006; Rab39b-rescue versus shRab39b P =0.01) subunits. The number of cells belongs from a minimum of three experimental replicates. * P

    Techniques Used: Expressing, Western Blot

    Related Articles

    Incubation:

    Article Title: N-Linked Glycosylation Is Required for Transferrin-Induced Stabilization of Transferrin Receptor 2, but Not for Transferrin Binding or Trafficking to the Cell Surface
    Article Snippet: .. Five micrograms of lysate was incubated with PNGase F or Endo Hf (New England Biolabs) according to the manufacturer’s protocol before Western analysis. .. To examine the binding of iron-loaded Tf (holo-Tf) to hTfR2, Hep3B cells were transiently transfected with wild-type or mutant hTfR2.

    Article Title: Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis *Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis * S⃞
    Article Snippet: .. Protein was deglycosylated by incubation with Endo Hf (New England Biolabs, 26,500 units per mg of pro-TPP1) at 25 °C for 15 h followed by 1 h at 37 °C. .. The solution was buffer exchanged and repurified by Mono Q12 chromatography as described above.

    Article Title: Evolutionarily conserved glycan signal to degrade aberrant brassinosteroid receptors in Arabidopsis
    Article Snippet: These plasmids were fully sequenced to ensure no PCR error and were individually transformed into YG414 or YG415 via a rapid transformation protocol ( ). .. Arabidopsis seedlings harvested from agar, soil, or liquid 1/2 Murashige and Skoog (MS) medium supplemented with or without BL (Chemiclones) or Kif (Toronto Research Chemicals) were ground in liquid N2 , dissolved (50 mg seedlings/100 μL) in 2× SDS buffer [0.125 M Tris pH 6.8, 4% (wt/vol) SDS, 20% (vol/vol) glycerol, 0.2 M DTT, 0.02% (wt/vol) bromophenol blue] and boiled for 10 min. After centrifugation, supernatants were used for immunoblot analyses or incubated with or without 1,000 U of Endo Hf in 1× G5 buffer (New England Biolabs) for 1 h at 37 °C. .. To perform the CHX chase experiment, 3-wk-old seedlings were transferred from 1/2 MS agar plates into 1/2 MS medium containing 180 μM CHX (Sigma), and equal amounts of seedlings were removed at different time points to extract total proteins into 2× SDS sample buffer.

    Western Blot:

    Article Title: N-Linked Glycosylation Is Required for Transferrin-Induced Stabilization of Transferrin Receptor 2, but Not for Transferrin Binding or Trafficking to the Cell Surface
    Article Snippet: .. Five micrograms of lysate was incubated with PNGase F or Endo Hf (New England Biolabs) according to the manufacturer’s protocol before Western analysis. .. To examine the binding of iron-loaded Tf (holo-Tf) to hTfR2, Hep3B cells were transiently transfected with wild-type or mutant hTfR2.

    Purification:

    Article Title: Evaluation of the contribution of the transmembrane region to the ectodomain conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein
    Article Snippet: Working dilutions for Western blotting were 1:2,000 goat anti-gp120 polyclonal antibody (ThermoFisher), 1:2,000 4E10 anti-gp41 antibody (Polymun Scientific, NIH AIDS Reagent Program), 1:10,000 mouse anti-β- actin (Abcam), 1:3,000 HRP-conjugated goat anti-human IgG (SantaCruz), 1:3,000 HRP-conjugated rabbit anti-goat IgG (ThermoFisher), and 1:10,000 HRP-conjugated goat anti-mouse IgG (ThermoScientific). .. Deglycosylation of Env glycoproteins Supernatants containing soluble glycoproteins or envelope proteins purified from cell membranes were denatured and treated with PNGase F or Endo Hf enzymes (New England BioLabs) for 1 ½ hours following the manufacturer’s protocol. ..

    Article Title: Improved production of recombinant human Fas ligand extracellular domain in Pichia pastoris: yield enhancement using disposable culture-bag and its application to site-specific chemical modifications
    Article Snippet: As for NFG1CG4-hFasLECD, the protein concentration of the desalted solution after the purification step with the Hi-Trap S 5 ml column was determined to be 9.9 mg/ml, and was directly used for the reaction with maleimide group containing compounds. .. Preparation of N-glycan trimmed tag-free hFasLECD Purified sample of tag-free hFasLECD was concentrated to 1.92 mg/ml with Amicon Ultra 15 (Molecular weight cut-off: 10 kDa), and digested with Endo Hf (New England Biolabs, Inc.). .. Twelve μl of 500 mM sodium citrate buffer (pH 5.5) was added to 192 μg of tag-free hFasLECD sample and then treated with 12000 U of Endo Hf at 37°C, for 48 h. The N-glycan trimmed tag-free hFasLECD in the reaction mixture was purified according to essentially the same procedures as described for NFG5-hFasLECD [ ].

    Mass Spectrometry:

    Article Title: Evolutionarily conserved glycan signal to degrade aberrant brassinosteroid receptors in Arabidopsis
    Article Snippet: These plasmids were fully sequenced to ensure no PCR error and were individually transformed into YG414 or YG415 via a rapid transformation protocol ( ). .. Arabidopsis seedlings harvested from agar, soil, or liquid 1/2 Murashige and Skoog (MS) medium supplemented with or without BL (Chemiclones) or Kif (Toronto Research Chemicals) were ground in liquid N2 , dissolved (50 mg seedlings/100 μL) in 2× SDS buffer [0.125 M Tris pH 6.8, 4% (wt/vol) SDS, 20% (vol/vol) glycerol, 0.2 M DTT, 0.02% (wt/vol) bromophenol blue] and boiled for 10 min. After centrifugation, supernatants were used for immunoblot analyses or incubated with or without 1,000 U of Endo Hf in 1× G5 buffer (New England Biolabs) for 1 h at 37 °C. .. To perform the CHX chase experiment, 3-wk-old seedlings were transferred from 1/2 MS agar plates into 1/2 MS medium containing 180 μM CHX (Sigma), and equal amounts of seedlings were removed at different time points to extract total proteins into 2× SDS sample buffer.

    Centrifugation:

    Article Title: Evolutionarily conserved glycan signal to degrade aberrant brassinosteroid receptors in Arabidopsis
    Article Snippet: These plasmids were fully sequenced to ensure no PCR error and were individually transformed into YG414 or YG415 via a rapid transformation protocol ( ). .. Arabidopsis seedlings harvested from agar, soil, or liquid 1/2 Murashige and Skoog (MS) medium supplemented with or without BL (Chemiclones) or Kif (Toronto Research Chemicals) were ground in liquid N2 , dissolved (50 mg seedlings/100 μL) in 2× SDS buffer [0.125 M Tris pH 6.8, 4% (wt/vol) SDS, 20% (vol/vol) glycerol, 0.2 M DTT, 0.02% (wt/vol) bromophenol blue] and boiled for 10 min. After centrifugation, supernatants were used for immunoblot analyses or incubated with or without 1,000 U of Endo Hf in 1× G5 buffer (New England Biolabs) for 1 h at 37 °C. .. To perform the CHX chase experiment, 3-wk-old seedlings were transferred from 1/2 MS agar plates into 1/2 MS medium containing 180 μM CHX (Sigma), and equal amounts of seedlings were removed at different time points to extract total proteins into 2× SDS sample buffer.

    Molecular Weight:

    Article Title: Improved production of recombinant human Fas ligand extracellular domain in Pichia pastoris: yield enhancement using disposable culture-bag and its application to site-specific chemical modifications
    Article Snippet: As for NFG1CG4-hFasLECD, the protein concentration of the desalted solution after the purification step with the Hi-Trap S 5 ml column was determined to be 9.9 mg/ml, and was directly used for the reaction with maleimide group containing compounds. .. Preparation of N-glycan trimmed tag-free hFasLECD Purified sample of tag-free hFasLECD was concentrated to 1.92 mg/ml with Amicon Ultra 15 (Molecular weight cut-off: 10 kDa), and digested with Endo Hf (New England Biolabs, Inc.). .. Twelve μl of 500 mM sodium citrate buffer (pH 5.5) was added to 192 μg of tag-free hFasLECD sample and then treated with 12000 U of Endo Hf at 37°C, for 48 h. The N-glycan trimmed tag-free hFasLECD in the reaction mixture was purified according to essentially the same procedures as described for NFG5-hFasLECD [ ].

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    New England Biolabs endoglycosidase hf
    Glycosidic processing analysis of WT and polymorphic α2 ARs. HEK293 cells were transfected with either WT or polymorphic HA-α2A or -α2C ARs. Forty-eight hours post-transfection, crude membranes were isolated and subjected to <t>endoglycosidase</t> digestion with no enzyme ( control ), endoglycosidase H f ( Endo H f ), or PNGase F ( PNGase ). Due to loss of signal during enzymatic digestion, 100 μg of protein was digested and loaded in each lane. Mature ( M ) and immature ( I ) glycosylated forms are indicated. Endo H cleaves only immature glycosylated forms, whereas PNGase cleaves all glycosylated forms. Molecular weight markers (kDa) are shown on the right. Note that HA-α2A WT and -α2A N251K ARs exhibit mostly mature (Endo H insensitive), whereas HA-α2C WT and -α2C Δ322-325 ARs show mostly immature (Endo H sensitive), glycosylation patterns, correlating with their predominant plasma membrane and intracellular localizations, respectively. The glycosylation pattern of neither polymorphic HA-α2 AR differed significantly with respect to its WT counterpart. Since α2 ARs are highly susceptible to degradation and aggregation, which can vary between experiments, attempts at quantification of any form of the receptor would not be reproducible and were not performed. Representative of three experiments
    Endoglycosidase Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/endoglycosidase hf/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    endoglycosidase hf - by Bioz Stars, 2021-03
    94/100 stars
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    Glycosidic processing analysis of WT and polymorphic α2 ARs. HEK293 cells were transfected with either WT or polymorphic HA-α2A or -α2C ARs. Forty-eight hours post-transfection, crude membranes were isolated and subjected to endoglycosidase digestion with no enzyme ( control ), endoglycosidase H f ( Endo H f ), or PNGase F ( PNGase ). Due to loss of signal during enzymatic digestion, 100 μg of protein was digested and loaded in each lane. Mature ( M ) and immature ( I ) glycosylated forms are indicated. Endo H cleaves only immature glycosylated forms, whereas PNGase cleaves all glycosylated forms. Molecular weight markers (kDa) are shown on the right. Note that HA-α2A WT and -α2A N251K ARs exhibit mostly mature (Endo H insensitive), whereas HA-α2C WT and -α2C Δ322-325 ARs show mostly immature (Endo H sensitive), glycosylation patterns, correlating with their predominant plasma membrane and intracellular localizations, respectively. The glycosylation pattern of neither polymorphic HA-α2 AR differed significantly with respect to its WT counterpart. Since α2 ARs are highly susceptible to degradation and aggregation, which can vary between experiments, attempts at quantification of any form of the receptor would not be reproducible and were not performed. Representative of three experiments

    Journal: Naunyn-Schmiedeberg's archives of pharmacology

    Article Title: Common α2A and α2C adrenergic receptor polymorphisms do not affect plasma membrane trafficking

    doi: 10.1007/s00210-014-0972-6

    Figure Lengend Snippet: Glycosidic processing analysis of WT and polymorphic α2 ARs. HEK293 cells were transfected with either WT or polymorphic HA-α2A or -α2C ARs. Forty-eight hours post-transfection, crude membranes were isolated and subjected to endoglycosidase digestion with no enzyme ( control ), endoglycosidase H f ( Endo H f ), or PNGase F ( PNGase ). Due to loss of signal during enzymatic digestion, 100 μg of protein was digested and loaded in each lane. Mature ( M ) and immature ( I ) glycosylated forms are indicated. Endo H cleaves only immature glycosylated forms, whereas PNGase cleaves all glycosylated forms. Molecular weight markers (kDa) are shown on the right. Note that HA-α2A WT and -α2A N251K ARs exhibit mostly mature (Endo H insensitive), whereas HA-α2C WT and -α2C Δ322-325 ARs show mostly immature (Endo H sensitive), glycosylation patterns, correlating with their predominant plasma membrane and intracellular localizations, respectively. The glycosylation pattern of neither polymorphic HA-α2 AR differed significantly with respect to its WT counterpart. Since α2 ARs are highly susceptible to degradation and aggregation, which can vary between experiments, attempts at quantification of any form of the receptor would not be reproducible and were not performed. Representative of three experiments

    Article Snippet: Membrane preparations (80–100 μg protein) were treated for 4 h with endoglycosidase Hf (Endo Hf ) or peptide- N -glycosidase (PNGase F) (New England Biolabs) per manufacturer's recommendations.

    Techniques: Transfection, Isolation, Molecular Weight

    Characterization of TM mod mutant Envs. a Cellular lysates and supernatants from 293T cells that were mock-transfected or transfected with TM mod Env DNAs were Western blotted. Western blots shown in this figure used goat anti-gp120 antibody or a mouse anti-β-actin control. The TM mod Envs with at least two new charged residues in the transmembrane region were secreted. The control TM mod 18 Env was inefficiently expressed and not secreted. The sgp140(−) Env lacks the transmembrane region. b The secreted Envs were analyzed by Blue Native PAGE. The sgp140(−) glycoprotein migrated as a heterogeneous mixture of monomers, dimers and trimers. The representative TM mod 10 protein migrated predominantly as a dimer. The TM mod 10v2 glycoprotein was also largely dimeric. Note that HIV-1 Envs migrate more slowly than expected in Blue Native gels. c Transfected cell supernatants containing sgp140(−) and TM mod 10 Envs were either mock-treated or treated with PNGase F (which removes all N-linked glycans) or Endo H f (which removes only high-mannose glycans). The Western blot shows that both sgp140(−) and TM mod 10 Envs resist Endo H f treatment, which indicates that they contain mostly complex carbohydrates. d Transfected cells expressing the E168K + N188A (EKNA) variant of TM mod 10, which allows HIV-1 JR-FL Envs to be recognized by the PG9 and PG16 neutralizing antibodies [ 96 – 99 ], were incubated with 50 mM kifunensine (a mannosidase I inhibitor). Cell supernatants were collected, deglycosylated with PNGase F or Endo H f, and Western blotted. Addition of kifunensine converted TM mod 10 glycosylation from mostly complex glycans to high-mannose glycans. e, f Cell supernatants containing the indicated soluble glycoproteins were precipitated with the indicated antibodies, and the precipitates were Western blotted with goat anti-gp120 antibody. The three soluble Envs exhibit a similar pattern of antigenicity. One-fourth volume of the supernatant used for immunoprecipitation was analyzed in the input lane. Data are representative of those obtained in at least two independent experiments

    Journal: Virology Journal

    Article Title: Evaluation of the contribution of the transmembrane region to the ectodomain conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein

    doi: 10.1186/s12985-017-0704-x

    Figure Lengend Snippet: Characterization of TM mod mutant Envs. a Cellular lysates and supernatants from 293T cells that were mock-transfected or transfected with TM mod Env DNAs were Western blotted. Western blots shown in this figure used goat anti-gp120 antibody or a mouse anti-β-actin control. The TM mod Envs with at least two new charged residues in the transmembrane region were secreted. The control TM mod 18 Env was inefficiently expressed and not secreted. The sgp140(−) Env lacks the transmembrane region. b The secreted Envs were analyzed by Blue Native PAGE. The sgp140(−) glycoprotein migrated as a heterogeneous mixture of monomers, dimers and trimers. The representative TM mod 10 protein migrated predominantly as a dimer. The TM mod 10v2 glycoprotein was also largely dimeric. Note that HIV-1 Envs migrate more slowly than expected in Blue Native gels. c Transfected cell supernatants containing sgp140(−) and TM mod 10 Envs were either mock-treated or treated with PNGase F (which removes all N-linked glycans) or Endo H f (which removes only high-mannose glycans). The Western blot shows that both sgp140(−) and TM mod 10 Envs resist Endo H f treatment, which indicates that they contain mostly complex carbohydrates. d Transfected cells expressing the E168K + N188A (EKNA) variant of TM mod 10, which allows HIV-1 JR-FL Envs to be recognized by the PG9 and PG16 neutralizing antibodies [ 96 – 99 ], were incubated with 50 mM kifunensine (a mannosidase I inhibitor). Cell supernatants were collected, deglycosylated with PNGase F or Endo H f, and Western blotted. Addition of kifunensine converted TM mod 10 glycosylation from mostly complex glycans to high-mannose glycans. e, f Cell supernatants containing the indicated soluble glycoproteins were precipitated with the indicated antibodies, and the precipitates were Western blotted with goat anti-gp120 antibody. The three soluble Envs exhibit a similar pattern of antigenicity. One-fourth volume of the supernatant used for immunoprecipitation was analyzed in the input lane. Data are representative of those obtained in at least two independent experiments

    Article Snippet: Deglycosylation of Env glycoproteins Supernatants containing soluble glycoproteins or envelope proteins purified from cell membranes were denatured and treated with PNGase F or Endo Hf enzymes (New England BioLabs) for 1 ½ hours following the manufacturer’s protocol.

    Techniques: Mutagenesis, Transfection, Western Blot, Blue Native PAGE, Expressing, Variant Assay, Incubation, Immunoprecipitation

    Effect of a fibritin trimerization motif on the TM mod 10 Env. a Cell lysates and supernatants from 293T cells expressing the EKNA variants of sgp140(−) or TM mod 10, or these Envs with a C-terminal fibritin trimerization domain (sgp140(−) FT and TM mod 10 FT, respectively), were analyzed on gels and Western blotted. A polyclonal goat anti-gp120 antibody was used to detect the Envs on the Western blots shown in this figure. Addition of the fibritin domain to the C-terminus of TM mod 10 did not prevent Env expression but significantly diminished release of the TM mod 10 FT glycoprotein from the cells. b The indicated soluble Envs were subjected to digestion with PNGase F or Endo H f and Western blotted. c Antibody precipitation of the EKNA variants of sgp140(−), sgp140(−) FT, TM mod 10 and TM mod 10 FT Envs secreted into the medium of expressing cells is shown. All EKNA Env constructs contain the E168K + N188A changes that restore the PG9/PG16 epitopes [ 96 – 99 ]. Data are representative of those obtained in duplicate or two independent experiments

    Journal: Virology Journal

    Article Title: Evaluation of the contribution of the transmembrane region to the ectodomain conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein

    doi: 10.1186/s12985-017-0704-x

    Figure Lengend Snippet: Effect of a fibritin trimerization motif on the TM mod 10 Env. a Cell lysates and supernatants from 293T cells expressing the EKNA variants of sgp140(−) or TM mod 10, or these Envs with a C-terminal fibritin trimerization domain (sgp140(−) FT and TM mod 10 FT, respectively), were analyzed on gels and Western blotted. A polyclonal goat anti-gp120 antibody was used to detect the Envs on the Western blots shown in this figure. Addition of the fibritin domain to the C-terminus of TM mod 10 did not prevent Env expression but significantly diminished release of the TM mod 10 FT glycoprotein from the cells. b The indicated soluble Envs were subjected to digestion with PNGase F or Endo H f and Western blotted. c Antibody precipitation of the EKNA variants of sgp140(−), sgp140(−) FT, TM mod 10 and TM mod 10 FT Envs secreted into the medium of expressing cells is shown. All EKNA Env constructs contain the E168K + N188A changes that restore the PG9/PG16 epitopes [ 96 – 99 ]. Data are representative of those obtained in duplicate or two independent experiments

    Article Snippet: Deglycosylation of Env glycoproteins Supernatants containing soluble glycoproteins or envelope proteins purified from cell membranes were denatured and treated with PNGase F or Endo Hf enzymes (New England BioLabs) for 1 ½ hours following the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Construct

    Effect of the C-terminal fibritin trimerization motif on membrane-anchored Envs. a Envs purified from transfected 293T cell membranes were analyzed by Blue Native PAGE. The Env(−)Δ712 FT and Env(+)Δ712 FT glycoproteins migrate at a size expected for trimers. b The purified soluble or membrane Envs were treated with PNGase F ( top panel ) or Endo H f ( bottom panel ). The arrows indicate Envs after Endo H f digestion. Compared to sgp140(−) and TM mod 10 Envs, which are relatively resistant to Endo H f digestion, purified membrane Envs are Endo H f -sensitive, and thus rich in high-mannose carbohydrates. c Flow cytometry was used to study Env surface expression and recognition by the 2G12 glycan-dependent antibody and the VRC01 CD4-binding site antibody. d To immunoprecipitate cell-surface Env, 293T cells transiently expressing the indicated Envs were incubated with antibodies, washed and lysed. Cell lysates were incubated with Protein A-Sepharose beads. Precipitates were Western blotted with a goat anti-gp120 antibody. e To assess cell-surface Env processing, cells expressing the indicated Envs were biotinylated as described in Methods. The cell lysates ( upper panel ) or deglycosylated cell-surface Envs ( lower panels ) were Western blotted with the 4E10 anti-gp41 antibody. After deglycosylation, the uncleaved Env is 75 kD, and the cleaved transmembrane Envs are 20–23 kD. f An α-complementation assay was used to measure Env-mediated cell-cell fusion. The reduced cell-cell fusion activity of Env(+)Δ712 FT was restored by the disruption of fibritin trimerization in the Env(+)Δ712 FT mut glycoprotein. g The infectivity of recombinant luciferase-expressing HIV-1 with the indicated Envs was measured on Cf2Th-CD4/CCR5 target cells. The luciferase activity in the target cells was normalized to that observed for the Env(+)Δ712 glycoprotein. h Virions pseudotyped with the indicated Env proteins were purified through a 20% sucrose cushion, denatured and Western blotted. Data in this figure are representative of, or averaged from, those obtained in at least two independent experiments. Error bars are standard deviations

    Journal: Virology Journal

    Article Title: Evaluation of the contribution of the transmembrane region to the ectodomain conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein

    doi: 10.1186/s12985-017-0704-x

    Figure Lengend Snippet: Effect of the C-terminal fibritin trimerization motif on membrane-anchored Envs. a Envs purified from transfected 293T cell membranes were analyzed by Blue Native PAGE. The Env(−)Δ712 FT and Env(+)Δ712 FT glycoproteins migrate at a size expected for trimers. b The purified soluble or membrane Envs were treated with PNGase F ( top panel ) or Endo H f ( bottom panel ). The arrows indicate Envs after Endo H f digestion. Compared to sgp140(−) and TM mod 10 Envs, which are relatively resistant to Endo H f digestion, purified membrane Envs are Endo H f -sensitive, and thus rich in high-mannose carbohydrates. c Flow cytometry was used to study Env surface expression and recognition by the 2G12 glycan-dependent antibody and the VRC01 CD4-binding site antibody. d To immunoprecipitate cell-surface Env, 293T cells transiently expressing the indicated Envs were incubated with antibodies, washed and lysed. Cell lysates were incubated with Protein A-Sepharose beads. Precipitates were Western blotted with a goat anti-gp120 antibody. e To assess cell-surface Env processing, cells expressing the indicated Envs were biotinylated as described in Methods. The cell lysates ( upper panel ) or deglycosylated cell-surface Envs ( lower panels ) were Western blotted with the 4E10 anti-gp41 antibody. After deglycosylation, the uncleaved Env is 75 kD, and the cleaved transmembrane Envs are 20–23 kD. f An α-complementation assay was used to measure Env-mediated cell-cell fusion. The reduced cell-cell fusion activity of Env(+)Δ712 FT was restored by the disruption of fibritin trimerization in the Env(+)Δ712 FT mut glycoprotein. g The infectivity of recombinant luciferase-expressing HIV-1 with the indicated Envs was measured on Cf2Th-CD4/CCR5 target cells. The luciferase activity in the target cells was normalized to that observed for the Env(+)Δ712 glycoprotein. h Virions pseudotyped with the indicated Env proteins were purified through a 20% sucrose cushion, denatured and Western blotted. Data in this figure are representative of, or averaged from, those obtained in at least two independent experiments. Error bars are standard deviations

    Article Snippet: Deglycosylation of Env glycoproteins Supernatants containing soluble glycoproteins or envelope proteins purified from cell membranes were denatured and treated with PNGase F or Endo Hf enzymes (New England BioLabs) for 1 ½ hours following the manufacturer’s protocol.

    Techniques: Purification, Transfection, Blue Native PAGE, Flow Cytometry, Cytometry, Expressing, Binding Assay, Incubation, Western Blot, Activity Assay, Infection, Recombinant, Luciferase

    (A) Endoglycosidase treatment of glycoproteins from secreted immature TBE virus from infected cells treated with 20 mM NH 4 Cl (lanes 1 to 4), secreted small immature subviral particles (prM mutant) (lanes 5 to 8), and secreted large immature subviral particles (prM mutant) (lanes 9 to 12). (B) Same treatment as described for panel A but using the corresponding mature forms. Samples were treated with 200 or 1,000 U of endo H f , as indicated, or 500 U of PNGase F (F) and compared to untreated controls (U) by SDS-PAGE and immunoblotting with polyclonal antiserum KVIII. The positions of the E and prM protein bands are indicated at the right.

    Journal: Journal of Virology

    Article Title: Two Distinct Size Classes of Immature and Mature Subviral Particles from Tick-Borne Encephalitis Virus

    doi: 10.1128/JVI.77.21.11357-11366.2003

    Figure Lengend Snippet: (A) Endoglycosidase treatment of glycoproteins from secreted immature TBE virus from infected cells treated with 20 mM NH 4 Cl (lanes 1 to 4), secreted small immature subviral particles (prM mutant) (lanes 5 to 8), and secreted large immature subviral particles (prM mutant) (lanes 9 to 12). (B) Same treatment as described for panel A but using the corresponding mature forms. Samples were treated with 200 or 1,000 U of endo H f , as indicated, or 500 U of PNGase F (F) and compared to untreated controls (U) by SDS-PAGE and immunoblotting with polyclonal antiserum KVIII. The positions of the E and prM protein bands are indicated at the right.

    Article Snippet: Endoglycosidase Hf (endo Hf ) and peptide N -glycosidase F (PNGase F) (New England Biolabs) were used according to the manufacturer's instructions with the provided buffers.

    Techniques: Infection, Mutagenesis, SDS Page

    Purification of N-glycan untrimmed and trimmed tag-free hFasLECDs. a) Elution profile of N-glycan untrimmed tag-free hFasLECD sample (pre-purified by 1st Hi-Trap S cation-exchange chromatography) in 2nd cation-exchange chromatography. Used column: Resource S 6 ml. The region shown in underbar was collected and used for characterization in c) . NaCl concentration under principal peak eluting condition is described. b) Elution profile of N-glycan trimmed tag-free hFasLECD sample in 3rd cation-exchange chromatography. Used column: Mono S 1 ml. The region shown in underbar was collected and used for characterization in c) . NaCl concentration under principal peak eluting condition is described. c) Elution profiles of fractionated products in size-exclusion chromatography. Solid line: N-glycan untrimmed tag-free hFasLECD. Dashed line: N-glycan trimmed tag-free hFasLECD. Used column: Superdex 200 10/300 GL. Elution buffer: 50 mM sodium acetate plus 150 mM NaCl (pH 5.6). Flow rate: 0.5 ml/min. The peak retention time of each sample is described. Vertical arrows indicate the elution positions of molecular-weight size-markers [Ald, aldolase (158 kDa); Ova, ovalbumin (44 kDa); Rna, ribonuclease A (13.7 kDa) under the same conditions. d) SDS-PAGE analysis of purification course during N-glycan trimming with Endo Hf glycosidase and receptor-mediated co-immunoprecipitation using wild type and mutant hFasRECD-T-Fcs. Lanes: a, N-glycan untrimmed tag-free hFasLECD; b, after Endo Hf digestion; c, after Con A column fractionation; d, after Mono S column fractionation; e, co-immunoprecipitated materials using wild-type hFasRECD-T-Fc [ 15 ]; f, co-immunoprecipitated materials using hFasRECD-T-Fc (N102Q, N120Q) mutant; M, Molecular-weight markers. “+CHO” and “ΔCHO” indicate the migration positions of N-glycan untrimmed and N-glycan trimmed tag-free hFasLECD, respectively.

    Journal: BMC Biotechnology

    Article Title: Improved production of recombinant human Fas ligand extracellular domain in Pichia pastoris: yield enhancement using disposable culture-bag and its application to site-specific chemical modifications

    doi: 10.1186/1472-6750-14-19

    Figure Lengend Snippet: Purification of N-glycan untrimmed and trimmed tag-free hFasLECDs. a) Elution profile of N-glycan untrimmed tag-free hFasLECD sample (pre-purified by 1st Hi-Trap S cation-exchange chromatography) in 2nd cation-exchange chromatography. Used column: Resource S 6 ml. The region shown in underbar was collected and used for characterization in c) . NaCl concentration under principal peak eluting condition is described. b) Elution profile of N-glycan trimmed tag-free hFasLECD sample in 3rd cation-exchange chromatography. Used column: Mono S 1 ml. The region shown in underbar was collected and used for characterization in c) . NaCl concentration under principal peak eluting condition is described. c) Elution profiles of fractionated products in size-exclusion chromatography. Solid line: N-glycan untrimmed tag-free hFasLECD. Dashed line: N-glycan trimmed tag-free hFasLECD. Used column: Superdex 200 10/300 GL. Elution buffer: 50 mM sodium acetate plus 150 mM NaCl (pH 5.6). Flow rate: 0.5 ml/min. The peak retention time of each sample is described. Vertical arrows indicate the elution positions of molecular-weight size-markers [Ald, aldolase (158 kDa); Ova, ovalbumin (44 kDa); Rna, ribonuclease A (13.7 kDa) under the same conditions. d) SDS-PAGE analysis of purification course during N-glycan trimming with Endo Hf glycosidase and receptor-mediated co-immunoprecipitation using wild type and mutant hFasRECD-T-Fcs. Lanes: a, N-glycan untrimmed tag-free hFasLECD; b, after Endo Hf digestion; c, after Con A column fractionation; d, after Mono S column fractionation; e, co-immunoprecipitated materials using wild-type hFasRECD-T-Fc [ 15 ]; f, co-immunoprecipitated materials using hFasRECD-T-Fc (N102Q, N120Q) mutant; M, Molecular-weight markers. “+CHO” and “ΔCHO” indicate the migration positions of N-glycan untrimmed and N-glycan trimmed tag-free hFasLECD, respectively.

    Article Snippet: Preparation of N-glycan trimmed tag-free hFasLECD Purified sample of tag-free hFasLECD was concentrated to 1.92 mg/ml with Amicon Ultra 15 (Molecular weight cut-off: 10 kDa), and digested with Endo Hf (New England Biolabs, Inc.).

    Techniques: Purification, Chromatography, Concentration Assay, Size-exclusion Chromatography, Flow Cytometry, Molecular Weight, SDS Page, Immunoprecipitation, Mutagenesis, Fractionation, Migration