Glycobuffer 3, by New England Biolabs - Product details

1

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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2

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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3

Glycosidase Digestion Analysis of Immunoprecipitated Proteins

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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.

Matsuoka K., Imahashi N., Ohno M., Ode H., Nakata Y., Kubota M., Sugimoto A., Imahashi M., Yokomaku Y, & Iwatani Y. (2022). SARS-CoV-2 accessory protein ORF8 is secreted extracellularly as a glycoprotein homodimer. The Journal of Biological Chemistry, 298(3), 101724.

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4

Glycoprotein Denaturation and Deglycosylation

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Protein samples (100 μg) were boiled in Glycoprotein Denaturing Buffer (NEB, Ipswich, MA, USA) for 10 min. For Endoglycosidase H (Endo H) treatment, samples were added to a reaction containing GlycoBuffer 3 (NEB) and Endo H (1000 units, NEB) and incubated at 37 °C for 1 h. For PNGase F treatment, samples were added to a reaction containing GlycoBuffer 2 (NEB), 1% Nonidet P-40 (NEB), and PNGase F (1000 units, NEB) and incubated at 37 °C for 1 h.

Nagata K., Takahashi M., Kiryu-Seo S., Kiyama H, & Saido T.C. (2017). Distinct functional consequences of ECEL1/DINE missense mutations in the pathogenesis of congenital contracture disorders. Acta Neuropathologica Communications, 5, 83.

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5

Protein Glycosylation Analysis Protocol

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Tissue was frozen and ground in microcentrifuge tubes with pestles for each genotype. For hae-3 hsl2-3 HSL2-YFP, hae-3 hsl2-9 er gl hae-3-YFP ebs3, and hae-3 hsl2-9 er gl hsl2-9-YFP ebs3 plants, two whole flowers (stage 16 and late stage 16) were ground and resuspended in 30 µl of SDS sample buffer. All samples were then boiled for ~3min and centrifuged to pellet tissue debris. Half of the supernatant of each sample was then treated with 1000U of Endo Hf with 1× Glyco Buffer 3 (NEB Cat. No. P0703S) for 1h at 37 °C (according to the manufacturer’s recommendations). Half of each sample was also left untreated. Samples were then separated by SDS–PAGE and immunoblotted as described below.

Baer J., Taylor I, & Walker J.C. (2016). Disrupting ER-associated protein degradation suppresses the abscission defect of a weak hae hsl2 mutant in Arabidopsis. Journal of Experimental Botany, 67(18), 5473-5484.

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6

Deglycosylation Analysis of Mutant SEPN1

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293TN cells transfected with pSelExpress-SEPN1^U428C were lysed in the buffer containing 150 mM NaCl, 20 mM HEPES pH 7.5, 10 mM EDTA and 1% Triton X100, and supplemented with protease inhibitors cocktail (Roche). Buffer exchange was performed on PD-10 column (GE Healthcare) using a gravity protocol as described by the manufacturer, and samples were eluted with PBS. A portion of the eluate (36 μL) containing 50 μg of total protein was supplemented with 4 μL of 10× glycoprotein denaturating buffer (New England BioLabs) and heated at 100 °C for 10 min. One-half of the resulting sample was then treated with recombinant endoglycosidase H (EndoH; New England BioLabs) with 1,000 U of enzyme in 30 μL of total reaction volume, supplemented with 10× GlycoBuffer 3 (New England BioLabs), for 1 h at 37 °C. A parallel control reaction was performed under the same conditions but without EndoH. The samples were then mixed with 4× Laemmli buffer and analyzed by Western blotting. Biological duplicates were performed for this assay.

Chernorudskiy A., Varone E., Colombo S.F., Fumagalli S., Cagnotto A., Cattaneo A., Briens M., Baltzinger M., Kuhn L., Bachi A., Berardi A., Salmona M., Musco G., Borgese N., Lescure A, & Zito E. (2020). Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity. Proceedings of the National Academy of Sciences of the United States of America, 117(35), 21288-21298.

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7

Glycosylation Analysis of HA-Fzo1

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To test if HA-Fzo1 is glycosylated, crude mitochondrial extracts of cells expressing endogenously HA-tagged Fzo1 were prepared. Extracts were treated with Endo H_f (NEB, P0703) in GlycoBuffer 3 (NEB) for 1 h at 37°C. HA-Fzo1 was eluted by adding Laemmli buffer and analysed by SDS–PAGE and immunoblotting.

Schuster R., Anton V., Simões T., Altin S., den Brave F., Hermanns T., Hospenthal M., Komander D., Dittmar G., Dohmen R.J, & Escobar-Henriques M. (2019). Dual role of a GTPase conformational switch for membrane fusion by mitofusin ubiquitylation. Life Science Alliance, 3(1), e201900476.

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8

Glycopeptidase F and Endo H Digestion

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For glycopeptidase F digestion, total protein lysates were extracted from infected cells following the Western blotting steps above. The protein was incubated for 10 min at 100°C, then incubated with 1% NP-40 and 1,000 U of glycopeptidase F (New England Biolabs, Beijing, China) in a total volume of 25 μl for 1 h at 37°C. For endoglycosides H (Endo H) digestion, the protein was adjusted to glycoprotein denaturing buffer and incubated for 10 min at 100°C, and we then added 10 × Glycobuffer 3 and Endo H (New England Biolabs) followed by incubation for 1 h at 37°C. The next steps were as described for the Western blotting.

Bu Y., Teng Q., Feng D., Sun L., Xue J, & Zhang G. (2021). YLMY Tyrosine Residue within the Cytoplasmic Tail of Newcastle Disease Virus Fusion Protein Regulates Its Surface Expression to Modulate Viral Budding and Pathogenicity. Microbiology Spectrum, 9(3), e02173-21.

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9

Glycoprotein Deglycosylation via EndoH Digestion

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For EndoH digestion, cell supernatants were concentrated by ultracentrifugation through a 20% (w/v) sucrose cushion (4 h at 100,000× g and 4 °C), and pellets were suspended in Tris-buffered saline TBS buffer (TBS; 50-mM Tris-HCl, pH 7.5, and 150-mM NaCl). Cells were lysed in TBS supplemented with 0.5% Nonidet P-40 (NP-40), and lysates were centrifuged for 5 min at 15,000× g and 4 °C. After the addition of 10 × glycoprotein denaturing buffer (New England Biolabs, Ipswich, MA, USA), samples were heated at 100 °C for 10 min. Next, samples were adjusted to 10 × GlycoBuffer 3 (New England Biolabs, Ipswich, MA, USA), divided in two portions and incubated for 1 h at 37 °C with or without 2-µL EndoH (New England Biolabs, Ipswich, MA, USA).

Zeyen L., Döring T, & Prange R. (2020). Hepatitis B Virus Exploits ERGIC-53 in Conjunction with COPII to Exit Cells. Cells, 9(8), 1889.

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10

In vitro Deglycosylation of Glycoproteins

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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.

Wang S., Cheng H., Wang L., Zhao R, & Guan D. (2020). Overexpression of NRF1-742 or NRF1-772 Reduces Arsenic-Induced Cytotoxicity and Apoptosis in Human HaCaT Keratinocytes. International Journal of Molecular Sciences, 21(6), 2014.

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Glycobuffer 3

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14 protocols using glycobuffer 3

Purification and Glycosylation Analysis of Recombinant Proteins

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

Glycosidase Digestion Analysis of Immunoprecipitated Proteins

Glycoprotein Denaturation and Deglycosylation

Protein Glycosylation Analysis Protocol

Deglycosylation Analysis of Mutant SEPN1

Glycosylation Analysis of HA-Fzo1

Glycopeptidase F and Endo H Digestion

Glycoprotein Deglycosylation via EndoH Digestion

In vitro Deglycosylation of Glycoproteins

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