Sequencing grade chymotrypsin

Manufactured by Roche

Sourced in Germany, United States

Sequencing grade chymotrypsin is a protease enzyme used in protein sequencing and analysis workflows. It specifically cleaves peptide bonds on the C-terminal side of large hydrophobic amino acid residues, such as tyrosine, tryptophan, and phenylalanine. This enzyme is purified to high purity and activity levels to ensure consistent and reliable performance in laboratory applications.

Automatically generated - may contain errors

Lab products found in correlation

Acrylamide bis solution, by Bio-Rad (2 mentions) Peptides α2ap wt 1 15 and α2ap q4s 1 15, by Biosynth (2 mentions) 4 20 precast tris glycine gels, by Bio-Rad (2 mentions) Dual color molecular weight standards, by Bio-Rad (2 mentions) Endoproteinase gluc, by Roche (2 mentions) Phe pro arg chloromethylketone ppack, by Prolytix (2 mentions) Human α2 antiplasmin, by Merck Group (2 mentions) Human plasma fibronectin, by Prolytix (2 mentions) Bovine thrombin, by Merck Group (2 mentions) Ferulic acid, by Merck Group (2 mentions) Glycine ethyl ester hydrochloride, by Merck Group (2 mentions) Waters synapt g2 s, by Waters Corporation (1 mentions) C18 ziptip, by Merck Group (1 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions) Gelcode blue stain reagent, by Thermo Fisher Scientific (1 mentions) Monodansylcadaverine mdc, by Merck Group (1 mentions) Thermo qe hf, by Thermo Fisher Scientific (1 mentions) Proteome discoverer, by Thermo Fisher Scientific (1 mentions) Ammonium bicarbonate, by Roche (1 mentions) Sequencing grade modified trypsin, by Roche (1 mentions)

8 protocols using sequencing grade chymotrypsin

Disulfide Bond Confirmation in rVHs

Check if the same lab product or an alternative is used in the 5 most similar protocols

For additional disulfide bond introduction, Gly or Ala and Ile at positions 54 and 78 of rVHs were both mutated to Cys by site-directed mutagenesis. Cys introduced mutants were prepared by the same methods as wild type rVHs, and disulfide formations were confirmed for H2-1-1, H3-9, and H3-15 as follows. rVHs and their mutants were treated with sequencing grade chymotrypsin (Roche, Basel, Switzerland) at 37 °C for 4 hours followed by addition of 10% formic acid, and then subjected to LC-MS analysis using a Waters Synapt G2S (Waters Corporation, Milford, MA, USA). Peptide identification was conducted with MassLynx Mass Spectrometry Software ver. 4.1 and BiopharmaLynx Software ver. 1.3 (Waters Corporation).

Shinozaki N., Hashimoto R., Fukui K, & Uchiyama S. (2017). Efficient generation of single domain antibodies with high affinities and enhanced thermal stabilities. Scientific Reports, 7, 5794.

+ Open protocol

+ Expand

Keap1 Protein Modifications by DATS

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human recombinant Keap1 protein was incubated at 37°C for 0.5 h with DATS (100 µM) or vehicle and was separated by NuPAGE® 4–12% bis-Tris Gel (Invitrogen, Carlsbad, CA, USA). After separation, the gel was stained with GelCode® Blue Stain Reagent (Thermo scientific, Rockford, IL, USA), and the gel pieces containing proteins were destained with 50% acetonitrile (ACN) containing 50 mM NH₄HCO₃ and vortexed until Coomassie brilliant blue (CBB) was completely removed. These gel pieces were then dehydrated in 100% ACN and vacuum-dried for 20 min with SpeedVac®. For the digestion, gel pieces were alkylated using 55 mM iodoacetamide in 50 mM NH₄HCO₃ for 0.5 h in dark. Finally, each gel piece was treated with 10 ng/µl sequencing grade chymotrypsin (Roche, Mannheim, Germany) in digestion buffer (100 mM Tris-HCl, 10 mM CaCl₂, pH 7.8) at 25°C for overnight. Following digestion, peptide fragments were extracted with 5% formic acid in 50% ACN solution at room temperature for 20 min. Supernatants were collected and dried with SpeedVac®. Samples resuspended in 0.1% formic acid were purified and concentrated using C18 ZipTips (Millipore, Billerica, MA, USA) before mass spectroscopic (MS) analysis.

Kim S., Lee H.G., Park S.A., Kundu J.K., Keum Y.S., Cha Y.N., Na H.K, & Surh Y.J. (2014). Keap1 Cysteine 288 as a Potential Target for Diallyl Trisulfide-Induced Nrf2 Activation. PLoS ONE, 9(1), e85984.

+ Open protocol

+ Expand

Chymotryptic Digestion and Mass Spectrometry of Cif Proteins

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Coomassie-stained SDS-PAGE gel bands corresponding to Cif-WT and Cif-E153Q proteins were destained to clarity in 50 mM ammonium bicarbonate/40% acetonitrile solution, dehydrated, and subjected to chymotryptic proteolysis using sequencing grade chymotrypsin (Roche Life Sciences; 15 ng/μl) overnight at room temperature. The resulting peptide digests were collected by extraction and dried by vacuum centrifugation, then loaded on to an in-house fabricated microcapillary HPLC column and resolved across a 50-minute gradient from 3% acetonitrile/0.125% formic acid to 35% acetonitrile/0.125% formic acid for online LTQ-Orbitrap data-dependent analysis essentially as described using a “top-10” method (21 (link)–23 (link)). The resulting MS/MS scans were data-searched using a modified Sequest algorithm (24 (link)) with no enzyme specificity against a single-protein FASTA containing the Cif-WT and Cif-E153Q sequences. Ion chromatograms were extracted to a mass measurement accuracy of +/− 1.5 parts-per-million from the calculated theoretical exact masses (http://physics.nist.gov). Annotated MS/MS spectral peaks were considered as matches within a 1 m/z tolerance from theoretical average masses.

Bahl C.D., Hvorecny K.L., Morisseau C., Gerber S.A, & Madden D.R. (2016). Visualizing the mechanism of epoxide hydrolysis by the bacterial virulence enzyme Cif. Biochemistry, 55(5), 788-797.

+ Open protocol

+ Expand

Recombinant FXIII-A2 and Thrombin Activity

Check if the same lab product or an alternative is used in the 5 most similar protocols

Recombinant human FXIII-A₂ was obtained from the late Dr. Paul Bishop (Zymogenetics, Seattle, WA), and recombinant human thrombin from E. Di Cera and L. Pelc (St. Louis University, MO). The activity of the recombinant human thrombin was determined using titration against hirudin.^{31 (link)} Human plasma fibronectin and Phe-Pro-Arg-chloromethyl ketone (PPACK) were supplied by Haematologic Technologies (Essex Junction, VT, USA). Bovine thrombin, human α₂-antiplasmin, actin, ferulic acid, α-cyano-4-hydroxycinnamic acid (α-CHCA), N,N-Dimethyl-p-phenylenediamine (DMPDA), monodansylcadaverine (MDC), and Glycine ethyl ester (GEE) hydrochloride were obtained from Millipore Sigma (St. Louis, MO, USA). Peptides α₂AP WT (1 – 15) and α₂AP Q4S (1 – 15) were synthesized by New England Peptide (Gardner, MA, USA). Peptide stock concentrations were determined by quantitative amino acid analysis (AAA Service Laboratory, Damascus, OR, USA). Sequencing grade chymotrypsin and GluC endoproteinase, both from Roche, were supplied by Millipore Sigma (St. Louis, MO, USA). 30% Acrylamide/Bis solution, 4 – 20 % precast tris-glycine gels, and dual-color molecular weight standards were purchased from Bio-Rad Laboratories (Richmond, CA, USA).

Syed Mohammed R.D., Ablan F.D., McCann N.M., Hindi M.M, & Maurer M.C. (2022). Transglutaminase Activities of Blood Coagulant Factor XIII are Dependent on the Activation Pathways and on the Substrates. Thrombosis and haemostasis, 123(4), 380-392.

+ Open protocol

+ Expand

Chymotrypsin Cleavage of AtMC4 Protein

Check if the same lab product or an alternative is used in the 5 most similar protocols

Wild-type AtMC4 crystals were harvested in stabilization buffer containing 100 mM sodium cacodylate, pH 6.4, 2.1 M ammonium sulfate. After washing five times with the stabilization buffer, crystals were dissolved in a cleavage reaction solution containing 25 mM HEPES, pH 7.6, 250 mM NaCl, 0.2 mM CaCl₂ for 10 min at room temperature. The cleaved AtMC4 fragments were further treated using sequencing-grade chymotrypsin (Roche, Inc.) following manufacturer’s manual. Different from AtMC4 that cleaves itself at a K or R position, chymotrypsin cleaves at an F, Y, or W position. Digested AtMC4 peptides was used for mass spectrometry analysis using a Thermo QE-HF (ThermoFisher) at the Stony Brook University Biological Mass Spectrometry Shared Resource. Data were processed using program Proteome Discoverer (ThermoFisher). The cleaved peptides and their positions are listed in Supplementary Table 2.

Zhu P., Yu X.H., Wang C., Zhang Q., Liu W., McSweeney S., Shanklin J., Lam E, & Liu Q. (2020). Structural basis for Ca2+-dependent activation of a plant metacaspase. Nature Communications, 11, 2249.

+ Open protocol

+ Expand

Botulinum Neurotoxin Proteolytic Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Botulinum neurotoxin is highly toxic and requires appropriate safety measures. All neurotoxins were handled in a class 2 biosafety cabinet equipped with HEPA filters. Commercially purified BoNT/G complex toxin was purchased (Metabiologics, Madison, WI). Sequencing-grade modified trypsin at 0.5 mg/mL in 50 mM acetic acid and sequencing grade chymotrypsin at 1 μg/μL in 50 mM ammonium bicarbonate was purchased (Roche, Pleasanton, CA). All chemicals were from Sigma-Aldrich (St. Louis, MO) except where indicated.

Kalb S.R., Baudys J, & Barr J.R. (2015). Detection of the HA-33 protein in botulinum neurotoxin type G complex by mass spectrometry. BMC Microbiology, 15, 227.

+ Open protocol

+ Expand

Acetylation Assay Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Nε-acetyl-lysine (AcK) was purchased from Chem-Impex International (Wood Dale, IL). Diaphorase (#D5540), resazurin (#199303), NAD⁺ (#N7004), NADP⁺ (#N5755), G6P (#G7879), DSS (#S1885), CHX (#C7698), SAHA (#SML0061), NAM (#72340), MG132 (#M7449), phenylmethanesulfonyl fluoride (#P7626), sodium fluoride (#S1504), sodium orthovanadate (#S6508), ATP (#A2383), and 1 M manganese(II) chloride solution (#M1787) were obtained from Sigma-Aldrich. Sequencing grade chymotrypsin (#11418467001) and DNase I (#10104159001) were purchased from Roche Diagnostics (Mannheim, Germany). InstantBlue Coomassie Protein Stain (#ab119211) was purchased from Abcam (Cambridge, UK). Protein quantification was performed with QPRO-BCA Kit Standard from Cyanagen (Bologna, Italy). HisPur Ni-NTA Resin (#TS-88222) was purchased from Thermo Scientific (Waltham, MA). Alexa Fluor 647-conjugated Annexin V (#A23204) was purchased from Invitrogen (Waltham, MA). Aprotinin (#616370), Leupeptin (#108975), Pepstatin A (#516481), and NADPH (#481973) were obtained from EMD Millipore. DNA oligomers were obtained from Sigma-Aldrich unless otherwise stated. Enzymes and buffers for molecular biology were purchased from NEB (Ipswich, MA, USA).

Wu F., Muskat N.H., Dvilansky I., Koren O., Shahar A., Gazit R., Elia N, & Arbely E. (2023). Acetylation-dependent coupling between G6PD activity and apoptotic signaling. Nature Communications, 14, 6208.

+ Open protocol

+ Expand

Recombinant Protein Characterization for Coagulation Research

Check if the same lab product or an alternative is used in the 5 most similar protocols

Recombinant human FXIII-A 2 was obtained from the late Dr. Paul Bishop (Zymogenetics, Seattle, WA), and recombinant human thrombin from E. Di Cera and L. Pelc (St. Louis University, MO). The activity of the recombinant human thrombin was determined using titration against hirudin. 31 Human plasma fibronectin and Phe-Pro-Arg-chloromethyl ketone (PPACK) were supplied by Haematologic Technologies (Essex Junction, VT, USA). Bovine thrombin, human α 2 -antiplasmin, actin, ferulic acid, α-cyano-4-hydroxycinnamic acid (α-CHCA), N,N-Dimethyl-p-phenylenediamine (DMPDA), monodansylcadaverine (MDC), and Glycine ethyl ester (GEE) hydrochloride were obtained from Millipore Sigma (St. Louis, MO, USA). Peptides α 2 AP WT (1 -15) and α 2 AP Q4S (1 -15) were synthesized by New England Peptide (Gardner, MA, USA). Peptide stock concentrations were determined by quantitative amino acid analysis (AAA Service Laboratory, Damascus, OR, USA). Sequencing grade chymotrypsin and GluC endoproteinase, both from Roche, were supplied by Millipore Sigma (St. Louis, MO, USA). 30% Acrylamide/Bis solution, 4 -20 % precast tris-glycine gels, and dual-color molecular weight standards were purchased from Bio-Rad Laboratories (Richmond, CA, USA).

Mohammed R.D.S., Ablan F.D.O., McCann N.M., Hindi M.M, & Maurer M.C. (2023). Transglutaminase Activities of Blood Coagulant Factor XIII Are Dependent on the Activation Pathways and on the Substrates. Thrombosis and haemostasis, 123(4).

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!