Trypsin ultra

Manufactured by New England Biolabs

Sourced in United States

Trypsin-ultra is a highly purified trypsin enzyme from bovine pancreas. Trypsin is a serine protease that cleaves peptide bonds on the carboxyl side of lysine and arginine residues. It is commonly used for the dissociation and disaggregation of adherent cells in cell culture applications.

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6 protocols using trypsin ultra

Influenza HA Digestion Assay

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In the TS assay, 5 μM H1/PR8 HA was pre-incubated separately with 25 μM of JNJ4796 and 10 μM CR6261 Fab for ~30 minutes at room temperature. Control reactions were incubated with 2% DMSO. The pH of each reaction was lowered using 1M sodium acetate buffer (pH 5.0). One reaction was retained at pH 7.4 to assess digestion at neutral pH. The reaction solutions were then thoroughly mixed and incubated for about 30 minutes at 37°C. After incubation, the reaction solutions were equilibrated at room temperature and the pH was neutralized by addition of 200 mM Tris buffer, pH 8.5. Trypsin-ultra^™ (NEB Inc.) was added at final ratio of 1:50 by mass and the samples were digested for about 40 minutes at 37°C. After incubation with trypsin, the reaction solutions were equilibrated at room temperature and quenched by addition of non-reducing SDS buffer and boiled for 2 minutes at 100°C. All samples were analyzed by 4–20% SDS-PAGE gel and imaged using BioRad ChemDoc^™ imaging system.

van Dongen M.J., Kadam R.U., Juraszek J., Lawson E., Brandenburg B., Schmitz F., Schepens W.B., Stoops B., van Diepen H.A., Jongeneelen M., Tang C., Vermond J., Real A.V., Blokland S., Garg D., Wu W., Goutier W., Lanckacker E., Klap J.M., Peeters D.C., Wu J., Buyck C., Jonckers T.H., Roymans D., Roevens P., Vogels R., Koudstaal W., Friesen R.H., Raboisson P., Dhanak D., Goudsmit J, & Wilson I.A. (2019). An orally active small molecule fusion inhibitor of influenza virus. Science (New York, N.Y.), 363(6431), eaar6221.

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Trypsin Susceptibility Assay for Influenza HA

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For the trypsin susceptibility assay, 0.5 μM HA was pre-incubated at pH 8.0 with 2.5 μM iHA-100, 1.0 μM 14A7, or 1.0 μM CR6261 for 30 min at room temperature^{18 (link)}. 14A7 is an HA head binding antibody produced in-house^{29 (link)}. CR6261 is an HA stalk binding antibody that protects HA from pH-induced proteases^{17 (link)}. Control reactions contained 2% DMSO without iHA-100. The pH of each reaction was lowered to pH 4.9 using 1 M sodium acetate buffer, while one reaction was retained at pH 8.0 to examine trypsin digestion at near-neutral pH. The reaction solutions were then thoroughly mixed by pipetting and incubated for 30 min at 37 °C. After incubation, the reaction solutions were neutralized to pH 8.0 with 1 M Tris-HCl buffer. Trypsin-ultra (New England Biolabs, Ipswich, MA) was added at a final ratio of 1:50 by mass, and the samples were digested for 20 min at 37 °C. Trypsin digestion was quenched by the addition of SDS loading buffer, and samples were boiled for 3 min at 99 °C. Finally, all samples were analyzed with 4–20% SDS-PAGE and immunoblotted with rabbit anti-H5HA antibody (Pep-10#11, produced in-house).

Saito M., Itoh Y., Yasui F., Munakata T., Yamane D., Ozawa M., Ito R., Katoh T., Ishigaki H., Nakayama M., Shichinohe S., Yamaji K., Yamamoto N., Ikejiri A., Honda T., Sanada T., Sakoda Y., Kida H., Le T.Q., Kawaoka Y., Ogasawara K., Tsukiyama-Kohara K., Suga H, & Kohara M. (2021). Macrocyclic peptides exhibit antiviral effects against influenza virus HA and prevent pneumonia in animal models. Nature Communications, 12, 2654.

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Trypsin Digestion of Recombinant H3-ATTH Protein

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A total of 0.4 μg recombinant H3-histidine (H3-ATTH) protein was incubated in the presence of 2.5 μg 3I14 or anti-SARS Fm-6 IgG1, or in the absence of antibody in Tris-HCl buffer at pH 8.0 containing 2 μg ml⁻¹ Trypsin-ultra (New England Biolabs, Ipswich, MA) at 37 °C. Trypsin digestion was stopped at several time-points by boiling the sample in a 100 °C water bath. Samples were run on 10% SDS–polyacrylamide electrophoresis gel under reducing conditions and blotted using a HisProbe-horseradish peroxidase and SuperSignal West HisProbe Kit (Pierce Biotechnology, Rockford, IL). Images have been cropped for presentation in Fig. 4a. Full-size images are presented in Supplementary Fig. 11. Data represent a representative experiment from three independent experiments.

Fu Y., Zhang Z., Sheehan J., Avnir Y., Ridenour C., Sachnik T., Sun J., Hossain M.J., Chen L.M., Zhu Q., Donis R.O, & Marasco W.A. (2016). A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve. Nature Communications, 7, 12780.

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HA Cleavage Assay with Inhibitors

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The assay was performed as previously described (20 (link)). Some 5-μM H1/PR8 HA were preincubated with 50 μM of P7 peptide, P7-TAMRA probe, or F0045 for 30 min at room temperature (control reactions consisted of a 2% DMSO vehicle). The pH of each reaction was lowered using 1-M sodium acetate buffer (pH 5.0). One reaction was retained at pH 7.4 to assess digestion at neutral pH. The reaction solutions were, then, thoroughly mixed and incubated for 20 min at 37 °C. The solutions were subsequently equilibrated to room temperature, and the pH was neutralized by addition of 200-mM Tris buffer, pH 8.5. Trypsin-ultra (NEB, Inc.) was added to all samples at a final ratio of 1:50 by mass, and the samples were digested for 30 min at 37 °C. After incubation with trypsin, the reactions were equilibrated to room temperature and quenched by addition of nonreducing SDS buffer and boiled for ∼2 min at 100 °C. All samples were analyzed by 4–20% SDS-PAGE gel and imaged using a BioRad ChemDoc imaging system.

Yao Y., Kadam R.U., Lee C.C., Woehl J.L., Wu N.C., Zhu X., Kitamura S., Wilson I.A, & Wolan D.W. (2020). An influenza A hemagglutinin small-molecule fusion inhibitor identified by a new high-throughput fluorescence polarization screen. Proceedings of the National Academy of Sciences of the United States of America, 117(31), 18431-18438.

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Cell Culture Reagents and Suppliers

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The drugs used in the present study were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless indicated otherwise: rapamycin (Cell Signaling Technology, Boston, MA, USA); plasmocin (Fisher Scientific, Hampton, NH, USA); EGF, dexamethasone, triiodothyronine, insulin–transferrin–selenium (ITS, 1X, Invitrogen, Carlsbad, CA, USA); penicillin/streptomycin (1X, Invitrogen); fetal bovine serum (Invitrogen); L-glutamine, HEPES, sodium pyruvate, and 2-mercaptoethanol. Trypsin-ultra, mass spectrometry-grade, restriction enzymes, and other molecular biology reagents were purchased from New England BioLabs (Ipswich, MA, USA).

Armando I., Cuevas S., Fan C., Kumar M., Izzi Z., Jose P.A, & Konkalmatt P.R. (2022). G Protein-Coupled Receptor 37L1 Modulates Epigenetic Changes in Human Renal Proximal Tubule Cells. International Journal of Molecular Sciences, 23(22), 14456.

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Enriching Phosphorylated Proteins from HCT116 Cells

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The cytosolic fraction of HCT116 cells in sodium phosphate buffer (50 mM, pH 7.0) at a concentration of 1.7 mg/mL was mixed with 1 mM of cPGA every three minutes for five times. Twenty minutes after final addition of cPGA solid urea was added to the sample to a final concentration of 8 M. Samples were reduced (5 mM DTT, 25 min, 56 °C), carbamidomethylated (15 mM IAA, 30 min, in the dark at room temperature), and diluted with 50 mM Tris–HCl to reach ≤5.5 M urea. Enzyme digestion was started by adding LysC (New England Biolabs, 1:50) and continued for twelve hours at 37 °C, followed by a five-hour incubation with trypsin-ultra (New England Biolabs, 1:50). The digested protein solution was adjusted to 0.1% TFA and 3 µg were desalted using a ZipTip-C₁₈ (Millipore) according to manufacturer’s instructions. Peptide eluates were dried in vacuum concentrator and dissolved in 0.1% TFA.

Akhmadi A., Yeskendir A., Dey N., Mussakhmetov A., Shatkenova Z., Kulyyassov A., Andreeva A, & Utepbergenov D. (2024). DJ-1 protects proteins from acylation by catalyzing the hydrolysis of highly reactive cyclic 3-phosphoglyceric anhydride. Nature Communications, 15, 2004.

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