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Endo hf

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Endo Hf is a type II restriction enzyme produced by New England Biolabs. It recognizes and cleaves the DNA sequence 5'-CGGT-3' and its reverse complement 5'-CCGC-3'.

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Lab products found in correlation

Pngase f, by New England Biolabs (21 mentions) Glycobuffer 3, by New England Biolabs (4 mentions) Sonicated, by Qsonica (3 mentions) Vivaspin 30 kda mwco filters, by Sartorius (3 mentions) Nebexpress ni spin columns, by New England Biolabs (3 mentions) His tag antibody, by Thermo Fisher Scientific (3 mentions) Pngase f, by Merck Group (3 mentions) Mini protean tgx stain free precast gel, by Bio-Rad (3 mentions) Endo h, by New England Biolabs (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Cellulase, by Merck Group (2 mentions) Ha from influenza a h1n1, by Sino Biological (2 mentions) Influenza a h3n2, by Sino Biological (2 mentions) Influenza a h5n1, by Sino Biological (2 mentions) Neuraminidase from influenza a h5n1, by Sino Biological (2 mentions) Hemagglutinin ha from influenza a h1n1, by New England Biolabs (2 mentions) Influenza a h1n1, by Sino Biological (2 mentions) Image studio 3, by LI COR (2 mentions) Odyssey imaging system, by LI COR (2 mentions) Nupage novex 4 12 bis tris protein gel, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Endo H HF (3 citations) Endoglycosidase Hf (Endo Hf) (2 citations)

83 protocols using endo hf

1

SARS-CoV-2 S Glycoprotein Deglycosylation

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SARS-CoV-2 S glycoproteins in cell lysates or on pseudovirus particles were prepared as described above. Protein samples were boiled in 1 × denaturing buffer and incubated with PNGase F or Endo Hf (New England Biolabs, Ipswich, MA) for 1 h at 37°C according to the manufacturer’s protocol. The samples were then analyzed by SDS-PAGE and Western blotting as described above.
Authentic SARS-CoV-2 viruses were obtained from BEI Resources (NIAID, NIH) and propagated for one passage on Vero-E6 cells. To prepare viral particles, cell supernatants were collected, centrifuged at low speed (2000 rpm, 10 min) to remove cell debris, and pelleted at 18,000 x g for 1 h at 4°C. Viral particles then were treated with 1% Triton X-100 and incubated with PNGase F or Endo Hf (New England Biolabs, Ipswich, MA) for 1 h at 37°C according to the manufacturer’s protocol. Virions were analyzed by Western blotting with the following primary antibodies: rabbit anti-SARS-Spike S1 (Sino Biological, Cat# 40591-T62), rabbit anti-SARS-Spike S2 (Sino Biological, Cat# 40590-T62), and SARS-CoV-2 Nucleocapsid Protein Monoclonal Antibody (CliniSciences, Cat# bsm-41414M). The Western blots were developed with the following secondary antibodies: HRP-conjugated anti-rabbit antibody (Cytiva, Marlborough, MA (NA934-1ML)) or HRP-conjugated goat anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA (Cat# 115-035-008)).
Wang Q., Nair M.S., Anang S., Zhang S., Nguyen H., Huang Y., Liu L., Ho D.D, & Sodroski J.G. (2021). Functional differences among the spike glycoproteins of multiple emerging severe acute respiratory syndrome coronavirus 2 variants of concern. iScience, 24(11), 103393.
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2

Deglycosylation of TconTS1 Enzyme

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About 2 mg of TconTS1 were incubated with 40,000 units of EndoHf (New England Biolabs) for 16 h at 37 °C in 2.0 ml 10 mM phosphate buffer (pH 7.4). The EndoHf-treated enzyme is termed H-TconTS1. A TconTS1 control without EndoHf addition was performed in parallel (TconTS1). The H-TconTS1 sample of replicate 1 was purified again using Strep-Tactin sepharose to remove free glycans and EndoHf, and buffer was exchanged to 10 mM phosphate buffer (pH 7.4) as already described. In contrast, the H-TconTS1 sample of replicate 2 was purified by three chromatography steps: (i) a PD-10 desalting column (Cytiva) to remove free glycans, (ii) by AffiSep ConA adsorbent (Galab) to remove remaining proteins with high-mannose type N-glycans, and (iii) finally treated by an amylose affinity purification (New England Biolabs) to remove EndoHf by its fused maltose-binding protein. All chromatography steps have been performed according to the manufacturer’s protocols. Finally, the buffer was exchanged again to 10 mM phosphate buffer (pH 7.4).
Rosenau J., Grothaus I.L., Yang Y., Kumar N.D., Ciacchi L.C., Kelm S, & Waespy M. (2022). N-glycosylation modulates enzymatic activity of Trypanosoma congolense trans-sialidase. The Journal of Biological Chemistry, 298(10), 102403.
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3

Enzymatic Deglycosylation and Detection

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Endo Hf: Post-nuclear lysate or eluate was denatured by adding Laemmli sample buffer and incubated at RT for 30 min, after which Endo Hf (New England Biolabs, Bioké, Leiden, The Netherlands) was added and incubated at 37 °C for 1 h prior to immunoblotting.
PNGase F: Eluted immunoprecipitated proteins were deglycosylated by adding G7 Reaction Buffer (New England Biolabs) and Remove-iT PNGase F (New England Biolabs), followed by incubation at 37 °C for 2 h. Note: the molecular weight of PNGase F (approximately 36 kDa) is approximately the same as that of TMEM129. To prevent distortion of the apparent molecular size of TMEM129 and putative glycosylated forms to occur, we used a removable PNGase F. Chitin Magnetic Beads (New England Biolabs) were pre-washed two times in TBS, added to the deglycosylated sample and incubated for 10 min at RT. PNGase F was removed by pelleting the beads and recovering the supernatant. Isolated proteins were denatured by adding Laemmli sample buffer, and then subjected to SDS-PAGE.
van de Weijer M.L., van Muijlwijk G.H., Visser L.J., Costa A.I., Wiertz E.J, & Lebbink R.J. (2016). The E3 Ubiquitin Ligase TMEM129 Is a Tri-Spanning Transmembrane Protein. Viruses, 8(11), 309.
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4

Endo Hf Treatment of Receptor Lysates

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Receptors were transiently expressed in HEK 293 cells in 60 mm plates, and cell lysates were prepared as described above. Protein concentration of samples was determined with the DC (detergent-compatible) Bio-Rad (St-Laurent, Canada) protein assay. For Endo Hf experiments, volumes of lysates corresponding to 40 μg protein were denatured in glycoprotein denaturing buffer 1× (New England Biolabs) for 30 min at 37°C followed by the addition of glycobuffer 1× (New England Biolabs) and 10,000 U/ml of Endo Hf (New England Biolabs). Samples were incubated with the enzyme for 5 h at 37°C and analyzed by Western blot.
Génier S., Degrandmaison J., Moreau P., Labrecque P., Hébert T.E, & Parent J.L. (2016). Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex. Molecular Biology of the Cell, 27(24), 3800-3812.
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5

Membrane Integration of SP-C Mutants

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To determine the extent of membrane integration of the wild-type and L188Q mutant proteins, single N-linked glycosylation acceptor sites were engineered at defined locations. Transfected cells expressing these constructs were metabolically labeled and SP-C proteins were isolated by immunoprecipitation. Samples were split and treated with endoglycosidase H (Endo H) or Endo Hf (NEB, P0702, P0703) to remove glycans or incubated without Endo H as a control, according to the manufacturer’s instructions. Samples were run on SDS-PAGE and processed for phosphorimaging.
Pobre-Piza K.F., Mann M.J., Flory A.R, & Hendershot L.M. (2022). Mapping SP-C co-chaperone binding sites reveals molecular consequences of disease-causing mutations on protein maturation. Nature Communications, 13, 1821.
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6

Protein Extraction from Leaf Tissue

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Protein extraction from leaf tissue was performed in the presence of two volumes of extraction buffer [50 mM sodium citrate, pH 5.5; 5% SDS (w/v); 0.01% BSA (w/v); 150 mM NaCl; 2% (v/v) β-mercaptoethanol and 10 µL of protease inhibitor cocktail (Sigma-Aldrich)] per 300 mg of tissue sample. The tissue was mechanically disrupted and boiled for 10 min. The samples were then centrifuged at maximum speed for 30 min at 4 °C and the supernatant collected. For Endo-H assays (Endo-Hf, New England Biolabs), 5 µL of total protein extract (approximately 10 µg) was used in each reaction and the protocol provided by the supplier was followed.
Vieira V., Peixoto B., Costa M., Pereira S., Pissarra J, & Pereira C. (2019). N-Linked Glycosylation Modulates Golgi-Independent Vacuolar Sorting Mediated by the Plant Specific Insert. Plants, 8(9), 312.
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7

Purification and Glycosylation Analysis of Recombinant Proteins

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Example 4

The spent culture media (P. pastoris constructs) were buffer-exchanged in 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and concentrated ten-fold using Vivaspin 30 kDa MWCO filters (Sartorius). To assess the extent of glycosylation, the concentrated spent culture media were subject to Endo Hf (New England Biolabs) digestions under native conditions in the presence of 1× GlycoBuffer 3 (50 mM sodium acetate, pH 6.0).

FCE proteins (wild-type and N-glycan variants) were purified from the concentrated spent cultures using NEBExpress Ni Spin Columns (New England Biolabs). The columns were washed twice with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 5 mM imidazole then once with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 10 mM imidazole. The purified protein was eluted with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 500 mM imidazole.

To prepare cell lysates (K. lactis constructs), cells were resuspended in 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and sonicated (Qsonica). The soluble cell lysate was pre-cleared by centrifugation at 16000×g for 15 minutes at 4° C. The cell lysates and spent culture media were analyzed by SDS-PAGE on 4-20% polyacrylamide gel, followed by western blotting with a His-tag antibody (Thermo Fisher Scientific).

US11098295B1. FCE mRNA capping enzyme compositions, methods and kits (2021-08-24). New England Biolabs, Inc. [US]. Inventors: Mehul Ganatra [US], Siu-Hong Chan [US], Christopher H. Taron [US], G. B. Robb [US].
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8

Purification and Analysis of P. pastoris and K. lactis Proteins

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Example 4

The spent culture media (P. pastoris constructs) were buffer-exchanged in 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and concentrated ten-fold using Vivaspin 30 kDa MWCO filters (Sartorius). To assess the extent of glycosylation, the concentrated spent culture media were subject to Endo Hf (New England Biolabs) digestions under native conditions in the presence of 1× GlycoBuffer 3 (50 mM sodium acetate, pH 6.0).

FCE proteins (wild-type and N-glycan variants) were purified from the concentrated spent cultures using NEBExpress Ni Spin Columns (New England Biolabs). The columns were washed twice with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 5 mM imidazole then once with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 10 mM imidazole. The purified protein was eluted with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 500 mM imidazole.

To prepare cell lysates (K. lactis constructs), cells were resuspended in 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and sonicated (Qsonica). The soluble cell lysate was pre-cleared by centrifugation at 16000×g for 15 minutes at 4° C. The cell lysates and spent culture media were analyzed by SDS-PAGE on 4-20% polyacrylamide gel, followed by western blotting with a His-tag antibody (Thermo Fisher Scientific).

US11028379B1. FCE mRNA capping enzyme compositions, methods and kits (2021-06-08). New England Biolabs, Inc. [US]. Inventors: Mehul Ganatra [US], Siu-hong Chan [US], Christopher H. Taron [US], G. B. Robb [US].
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9

Recombinant Lipase Characterization via Western Blot

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The recombinant lipase was tested by Western blotting as described by Shen et al. [16 (link)]. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 5% stacking gel and 12% resolving gel. Monoclonal mouse-anti-His-Tag antibody (Tiangen, Beijing, China) was used as the primary antibody and goat anti-mouse IgG antibody conjugated to alkaline phosphatase (Sigma-Aldrich, St. Louis, Missouri, United States) as the secondary antibody. The membrane was applied to a BCIP/NBT Chromogenic reagent kit (Tiangen) according to the manufacturer’s instructions.
Protein concentration was quantified by Bradford assay [53 (link)] using Bovine Serum Albumin (BSA) as a standard. Glycosyl chains in pRML were removed by Endo Hf (New England Biolabs; Beverly, Massachusetts, United States) according to the manufacturer’s instructions.
Huang J., Xia J., Yang Z., Guan F., Cui D., Guan G., Jiang W, & Li Y. (2014). Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel. Biotechnology for Biofuels, 7, 111.
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10

Surface Biotinylation and Glycosidase Treatment

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Surface biotinylation was carried out essentially as in Hanus et al. (2016) (link). Dissociated neurons were washed in imaging buffer containing (in mM): 120 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 15 glucose, 10 HEPES pH 7.4. Cells were then treated with the same buffer containing NHS-SS-biotin (0.8 to 1 mg/mL, Thermo) at room temperature for 7 min. Cells were rinsed and excess biotinylating reagent was quenched using the same buffer supplemented with 10–20 mM L-lysine. Cells were lysed in PBS containing 1% triton X-100, 0.6%SDS and a protease inhibitor cocktail. Biotinylated proteins were purified from cell lysates over streptavidin-conjugated agarose beads and eluted by reduction of disulfide-linked biotin with 50 mM DTT for 15 min at 75 ˚C. Purified surface fractions were divided and either left untreated or treated with endoHf (New England Biolabs) or PNGase (New England Biolabs) according to the manufacturer’s insctructions. Extracts were diluted (~1.5 fold) in sodium phosphate (50 mM, pH 5.5 final) or sodium citrate buffer (50 mM pH 7.5) plus NP40 (or triton X-100, 1% final) for PNGase and endoH respectively. Enzymes were used at 1000 (PNGase) or 3000 (endoH) units/µg total protein and incubated overnight at 37 ˚C.
Bowen A.B., Bourke A.M., Hiester B.G., Hanus C, & Kennedy M.J. (2017). Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines. eLife, 6, e27362.
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