Trypsin

Manufactured by G Biosciences

Trypsin is a serine protease enzyme that is commonly used in laboratory settings to facilitate cell detachment and dissociation. Its primary function is to cleave peptide bonds, particularly those involving the carboxyl group of lysine or arginine residues. This enzymatic activity is essential for various cell culture applications, where trypsin is employed to release adherent cells from the growth surface, enabling their isolation, propagation, or further experimental manipulation.

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Lab products found in correlation

Atlantis c18 column, by Waters Corporation (4 mentions) Q tof api us quad tof mass spectrometer, by Waters Corporation (3 mentions) Nanoacquity uplc, by Waters Corporation (1 mentions) Fs30d bath sonicator, by Thermo Fisher Scientific (1 mentions) Sialidase, by Roche (1 mentions) Sialic acid quantification kit, by Merck Group (1 mentions) Aprotinin, by Thermo Fisher Scientific (1 mentions) Ammonium persulfate, by Thermo Fisher Scientific (1 mentions) Mineral oil, by Avantor (1 mentions) Dithiothreitol (dtt), by Avantor (1 mentions) Bovine serum albumin (bsa), by Avantor (1 mentions) Agarose 1, by Avantor (1 mentions) Vacutainer sst, by BD (1 mentions) 2 d sds page standards, by Bio-Rad (1 mentions) Acetonitrile, by Merck Group (1 mentions) Methanol, by Merck Group (1 mentions) Formic acid, by Merck Group (1 mentions) Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions) Tissuelyser 2, by Qiagen (1 mentions) Cem discover microwave digestor, by G Biosciences (1 mentions)

Close spelling variants of this product

Sequencing Grade Trypsin

9 protocols using trypsin

Worm Protein Extraction and Preparation

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The worm pellet was ground into fine powder using a single 2.8 mm i.d. steel ball with a TissueLyser II (Qiagen, Valencia, CA) at a frequency of 30 Hz for 30 sec, followed by the addition of 100 μl extraction solvent of PBS buffer, pH 7.5, with protease inhibitor and vortexing. Supernatants were collected after centrifugation at 16,000 × g (10 min, 4 °C). Protein was then quantified with a Qubit Protein Assay Kit (Life Technologies, Carlsbad, CA) in compliance with the manufacturer’s protocol. Disulfide bonds were reduced using 100 mM dithiothreitol (DTT) at a ratio of 1:10 DTT/sample volume and incubated at 50 °C for 45 min. Cysteine bonds were then alkylated with 200 mM iodoacetamide at the same volume ratio for 20 min at room temperature. Finally, protein was digested with trypsin (G-Biosciences, St. Louis, MO) at a 1:50 ratio of trypsin/protein, and incubated at 37 °C for 12 hr.

Liu Y., Sellegounder D, & Sun J. (2016). Neuronal GPCR OCTR-1 regulates innate immunity by controlling protein synthesis in Caenorhabditis elegans. Scientific Reports, 6, 36832.

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Mass Spectrometric Identification of BioR Proteins

Cited 1 time

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The identity of two versions of P. denitrificans BioR proteins (BioR1 and BioR2) was verified using A Waters Q-Tof API-US Quad-ToF mass spectrometer connected to a Waters nano Acquity UPLC (Feng and Cronan 2011 (link)). As we described before (Feng and Cronan 2011 (link)), the protein band of interest was digested with Trypsin (G-Biosciences St. Louis, MO), and the resultant peptides were loaded on a Waters Atlantis C-18 column (0.03 mm particle, 0.075 × 150 mm). The dependently acquired data were further subjected to the ms/ms analyses.

Feng Y., Kumar R., Ravcheev D.A, & Zhang H. (2015). Paracoccus denitrificans possesses two BioR homologs having a role in regulation of biotin metabolism. MicrobiologyOpen, 4(4), 644-659.

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Red Blood Cell-Coated Nano-Particles Formulation

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A series of RBC-NP formulations were prepared by coating 1 mg of PLGA cores with RBC membranes collected from 200 μL, 100 μL, 75 μL, 50 μL, 25 μL or 0 μL of mouse blood. Each formulation was then adjusted to 1 × PBS buffer (pH = 7.4) and sonicated for 5 min using an FS30D bath sonicator (Fisher Scientific, Waltham, MA) at a frequency of 42 kHz and a power of 100W to facilitate the aggregation process. After sonication, the hydrodynamic diameter of the particles was determined using DLS. trypsinized RBC-NPs were prepared by incubating a stable RBC-NP formulation (100 μL blood per mg PLGA core) with 50 μg mL⁻¹ trypsin (G-Biosciences, St. Louis, MO). Two hours following the trypsinization, the particle size was measured by DLS.

Luk B.T., Hu C.M., Fang R.H., Dehaini D., Carpenter C., Gao W, & Zhang L. (2014). Interfacial Interactions between Natural RBC Membranes and Synthetic Polymeric Nanoparticles. Nanoscale, 6(5), 2730-2737.

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Quantifying RBC-NP Glycoproteins and Sialic Acid

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RBC-NPs were first prepared with 100 nm PLGA cores and RBC membrane vesicles. For glycoprotein quantification, proteins exposed on the RBC-NP surfaces were trypsinized by incubating 1 mL of 1 mg mL⁻¹ RBC-NPs with 5 μg of trypsin (G-Biosciences, St. Louis, MO) at room temperature for 2 h. Sialic acid removal from RBC-NPs was performed by incubating 1 mL of 1 mg mL⁻¹ of RBC-NPs in water with 100 units of sialidase (Roche Diagnostics, Indianapolis, IN) at room temperature for 2 h. The samples were then centrifuged at 200,000 × g for 45 min using an Optima L-90K Ultracentrifuge (Beckman Coulter, Brea, CA), and the supernatant was collected and examined for glycoprotein and sialic acid content. To quantify glycoprotein, the supernatant was examined using Glycoprotein Detection Reagent (Thermo Scientific) following manufacturer’s instructions. To quantify sialic acid, the supernatant was examined using Sialic Acid Quantification Kit (Sigma-Aldrich, St. Louis, MO) following the manufacturer’s instructions. Equivalent amounts of RBC ghosts and bare PLGA cores were used as a positive and a negative control, respectively.

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Identifying Chimeric MCR Proteins

Cited 2 times

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The identities of the four chimeric proteins of MCR-1 and -2 (TM-MCR-1, TM1-EptA, TM-MCR-2, and TM2-EptA) were examined using a Waters quadrupole time of flight (QTOF) API-US mass spectrometer (53 (link), 54 (link)). A band obtained from SDS-PAGE separation of purified protein was digested with trypsin (G-Biosciences St. Louis, MO), and the resultant peptides were analyzed with a Waters Atlantis C₁₈ column (0.03-mm particle size, 0.075 by 150 mm). Finally, the acquired data were subjected to further analyses through the Waters ProteinLynx Global Server 2.2.5, Mascot (Matrix Sciences), and BLAST against the NCBI nr database.

Xu Y., Wei W., Lei S., Lin J., Srinivas S, & Feng Y. (2018). An Evolutionarily Conserved Mechanism for Intrinsic and Transferable Polymyxin Resistance. mBio, 9(2), e02317-17.

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Identifying S. suis BirA protein

Cited 2 times

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A Waters Q-Tof API-US Quad-ToF mass spectrometer was applied to determine the identity of S. suis BirA (BirA_ss) protein1 (link)46 (link). The purified protein band was cut from the gel and digested with Trypsin (G-Biosciences St. Louis, MO), giving a pool of overlapping peptides loaded on a Waters Atlantis C-18 column (0.03 mm particle, 0.075 mm × 150 mm). The acquired data were subjected to the ms/ms analyses.

Ye H., Cai M., Zhang H., Li Z., Wen R, & Feng Y. (2016). Functional definition of BirA suggests a biotin utilization pathway in the zoonotic pathogen Streptococcus suis. Scientific Reports, 6, 26479.

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Two-Dimensional Gel Electrophoresis Workflow

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Where applicable, consumables were of electrophoresis grade or higher. Vacutainer^® SST (serum-separator tubes) and 21G butterfly needles were from BD (Franklin Lakes, NJ, United States). ReadyStrip^TM immobilized pH gradient (IPG) strips (17 cm, pH 3–10 non-linear), Bio-Lyte carrier ampholytes (pH 3-10, pH 4-6), and 2-D SDS-PAGE Standards were from Bio-Rad Laboratories (Hercules, CA, United States). AEBSF, agarose I, bovine serum albumin (BSA), CHAPS, dithiothreitol (DTT), leupeptin, mineral oil, and TG-SDS buffer concentrate were from Amresco (Solon, OH, United States). Acetic acid was from Anachemia (Montreal, Quebec); sodium dodecyl sulfate (SDS) was from J. T. Baker Chemical Co. (Phillipsburg, NJ, United States); mass spectrometry-grade trypsin was from G-Biosciences (St. Louis, MO, United States); Coomassie Brilliant Blue G-250 (CBB) was from Genlantis (San Diego, CA, United States); and Broad-range (200–10 kDa) Unstained Protein Standard was from New England Biolabs (Ipswich, MA, United States). Ammonium persulfate and aprotinin were from Thermo Fisher Scientific (Waltham, MA, United States). Acetonitrile, formic acid, and methanol were from EMD Millipore (Burlington, MA, United States). Acrylamide/bis-acrylamide (37.5:1) solution and all other chemicals utilized were from Alfa Aesar (Haverhill, MA, United States). Double glass-distilled water (ddH₂O) was used throughout.

Kurgan N., Noaman N., Pergande M.R., Cologna S.M., Coorssen J.R, & Klentrou P. (2019). Changes to the Human Serum Proteome in Response to High Intensity Interval Exercise: A Sequential Top-Down Proteomic Analysis. Frontiers in Physiology, 10, 362.

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Proteomic Analysis of Trypanosome PolyP Proteins

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Samples containing T. brucei and T. cruzi polyP-binding proteins from two independent
experiments were processed at the Protein Sciences Facility of University of Illinois (Urbana, IL) for liquid chromatography
tandem mass spectrometry analysis (LC-MS/MS). Sample cleanup was performed using Perfect Focus according to manufacturer’s
instructions. Protein samples were reduced in 10 mM DTT at 56°C for 30 min and alkylation was performed using 20 mM
iodoacetamide for 30 min in the dark. Samples were digested with trypsin (G-Biosciences, St. Louis, MO) at a ratio of 1:10
– 1:50 using a CEM Discover Microwave Digestor (Mathews, SC) at 55˚C for 15 min. Digested peptides were extracted
with 50% acetonitrile, 5% formic acid, dried under vacuum and resuspended in 5% acetonitrile, 0.1% formic acid for LC-MS/MS
analysis.

Negreiros R.S., Lander N., Huang G., Cordeiro C.D., Smith S.A., Morrissey J.H, & Docampo R. (2018). Inorganic Polyphosphate Interacts with Nucleolar and Glycosomal Proteins in Trypanosomatids. Molecular microbiology, 110(6), 973-994.

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Proteomic Identification of BirA Orthologues

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A Waters Q-Tof API-US Quad-ToF mass spectrometer was used for the determination of the two L. lactis BirA orthologues (BirA1_{_LL} & BirA2_{_LL}). The protein band of interest was removed from the gel and digested with Trypsin (G-Biosciences St. Louis, MO). Finally, the resultant peptides were loaded on a Waters Atlantis C-18 column (0.03 mm particle, 0.075 mm × 150 mm) and the acquired data were subjected for further analyses by the ms/ms.

Zhang H., Wang Q., Fisher D.J., Cai M., Chakravartty V., Ye H., Li P., Solbiati J.O, & Feng Y. (2016). Deciphering a unique biotin scavenging pathway with redundant genes in the probiotic bacterium Lactococcus lactis. Scientific Reports, 6, 25680.

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