Dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate (ABC), ammonium acetate (AA), ammonium hydroxide, ribonuclease B from bovine pancreas (RNase B), fetuin from fetal calf serum (bovine fetuin), asialofetuin from fetal calf serum (asialofetuin) and alpha-1 acid glycoprotein from human plasma (AGP) were purchased from Sigma-Aldrich (St. Louis, MO). Murine IgG1 (Intact mAb Mass Check Standard) was obtained from Waters Corporation (Milford, MA). α2–3 neuraminidase, α2–3,6,8,9 neuraminidase, β1–3 galactosidase, β1–4 galactosidase and β1–3,4 galactosidase were acquired from New England Biolabs (Ipswich, MA). Mass spectrometry grade Trypsin/Lys-C mixture, sequencing grade chymotrypsin and sequencing grade endoprotease Glu-C (Glu-C) were obtained from Promega (Madison, WI). HPLC grade water was acquired from Mallinckrodt Chemicals (Phillipsburg, NJ). HPLC grade acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ).
Sequencing grade chymotrypsin
Manufactured by Promega
Sourced in United States
Sequencing grade chymotrypsin is a proteolytic enzyme used in protein sequencing applications. It cleaves peptide bonds on the carboxyl side of aromatic amino acid residues, such as tyrosine, tryptophan, and phenylalanine, generating peptide fragments for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Iodoacetamide, by Merck Group (3 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Asialofetuin, by Merck Group (1 mentions)
Asialofetuin from fetal calf serum, by Merck Group (1 mentions)
α2 3 neuraminidase, by New England Biolabs (1 mentions)
β1 4 galactosidase, by New England Biolabs (1 mentions)
Ribonuclease b from bovine pancreas, by Merck Group (1 mentions)
Rnase b, by Merck Group (1 mentions)
Alpha 1 acid glycoprotein from human plasma, by Merck Group (1 mentions)
α2 3 6 8 9 neuraminidase, by New England Biolabs (1 mentions)
β1 3 galactosidase, by New England Biolabs (1 mentions)
β1 3 4 galactosidase, by New England Biolabs (1 mentions)
Hplc grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Hplc grade water, by Mallinckrodt (1 mentions)
11 protocols using sequencing grade chymotrypsin
1
Glycoprotein Analysis: Enzymatic Approaches
2
Chymotrypsin-Assisted Oleosome Digestion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The most relevant protein bands, detected for the electrophoretic gel of oleosome samples before and after gastroduodenal digestion experiments, were excised from the gel and digested according to the protocol described by De Angelis et al. [16 (link)], with few modifications regarding the proteolytic enzyme selected. Specifically, trypsin was replaced by chymotrypsin, thus an appropriate buffer (Tris-HCl 100 mM, pH 8.0 + 10 mM CaCl2) was used to prepare dithiothreitol (10 mM stock solution) and iodoacetamide (55 mM stock solution) solutions. Moreover, a calculated amount of sequencing grade chymotrypsin (Promega Corporation, WI, USA. Source: bovine pancreas, activity: 70 units/mg by BTEE) was added to the sample, so that a ratio 1/20 enzyme/protein was attained to facilitate a complete enzymatic digestion. The digestion process was performed at 25 °C, while keeping samples shaking at 500 rpm with an Innova 4000 Benchtop Incubator Shaker (New Brunswick Scientific, NJ, USA), and it finally lasted 16 h. After drying, each digested sample was suspended in 80 µL of H2O/ACN 95/5 + 0.1% formic acid (v/v) and analyzed by mass spectrometry.
3
Biochemical Characterization of Purified Proteins
4
Monoclonal Antibodies for BoNT Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CR2 and RAZ1, monoclonal antibodies to BoNT/A, BoNT/B (2B18.2 and B12.1), and BoNT/E (4E17.1) were attained from Dr. James Marks of the University of California at San Francisco [21 (link),22 (link),23 ]. Polyclonal antibodies to BoNT/F1 were procured from Metabiologics (Madison, WI, USA). Dynabeads® (M-280/Streptavidin) were obtained from Invitrogen (Carlsbad, CA, USA). BoNT/A, /E, and /F complex toxins were purchased from Metabiologics (Madison, WI, USA). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. Sulfo-NHS-Biotin was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Kingfisher plates and tip combs were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Trypsin (gold, mass spectrometry grade) and sequencing grade chymotrypsin were purchased from Promega (Madison, WI, USA).
5
Protein Identification by Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For protein analysis by mass spectrometry, a Coomassie stained protein band was excised from a polyacrylamide gel and digested with sequencing grade chymotrypsin (Promega, Madison, WI) (39 (link)). Proteolytic fragments were analyzed by LC-MS/MS with electrospray ionization using a Thermofisher Hypersil Gold 80 Å reverse-phase column (Torrance, CA) and Exion UHPLC linked to a SCIEX 5600 Triple-Tof mass spectrometer (SCIEX, Toronto, Canada). The MS/MS data were processed to provide protein identifications using an in-house Protein Pilot 5.0 search engine (Sciex, Toronto, Canada) using the B. anthracis UniProt protein database and a chymotrypsin plus missed cleavage digestion parameter. Sequences identified in the software were verified by manual de novo sequencing for authenticity against the known sequences of BclA and/or BxpB proteins.
6
SARS-CoV-2 Protease Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serotonin (5-hydroxytryptamine) and dithiothreitol (DTT) were obtained from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). 5-Hydroxyindoleacetic acid (5HIAA) was procured from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Fremy's reagent (potassium nitrosodisulfonate) was purchased from Merck Sigma-Aldrich (MA, USA). AVLQSGFR (Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg) was obtained from GenScript Biotech Corp. AVLQ (Ala-Val-Leu-Gln) and stable AVLQ ([13C315N-Ala]-Val-Leu-Gln) were purchased from GL Biochem, Ltd. (Shanghai, China). Sequencing grade chymotrypsin was procured from Promega (WI, USA). Rabbit polyclonal antibodies to SARS-CoV-2 main protease (3C-like protease) were purchased from GeneTex Inc. (CA, USA). Anti-TD-modified protein monoclonal antibody (1B7) and Q5HIAA-modified protein monoclonal antibody (2B1) were prepared as previously described [10 (link),14 (link)]. Rabbit TrueBlot® (anti-rabbit IgG horseradish peroxidase (HRP)) was obtained from Rockland Immunochemicals, Inc. (PA, USA). Lipofectamine® 3000, Opti-MEM®, and sulfo–NHS–LC-biotin were purchased from Thermo Fisher Scientific (MA, USA). Unless otherwise indicated, all other chemicals used were of high quality. A recombinant SARS-CoV-2 main protease was previously prepared [8 (link)]. The plasmid (pLEX307-SARS-CoV-2-3CL WT, #160278) was obtained from Addgene [21 (link)].
7
Proteolysis Kinetics Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins (0.1 mg/ml) were reacted with sequencing grade chymotrypsin (Promega) at 1:50 (w/w) ratio or with subtilisin from Bacillus licheniformis (Sigma) at 1:1000 (w/w) ratio, in 10 mM Tris, 145 mM NaCl, 5 mM CaCl2, pH 7.4 at 37 °C, in the absence or presence of the active site inhibitor argatroban (500 μM). Aliquots of proteolysis mixtures were quenched whit NuPAGE LDS buffer and analyzed by non-reducing SDS-PAGE (4–12% acrylamide). None of the gels were cropped and originals are available in the Supplementary Information. The relative intensity of intact protein gel bands, after Coomassie staining, was estimated by densitometric analysis and fit to single exponential decay over time.
8
Membrane Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PrestoBlue® Cell Viability Reagent, Mem-PER™ Plus Membrane Protein Extraction Kit, and all reagents for cell culture were purchased from Life Technologies (Carlsbad, CA, USA). Bradford Protein Assay and Clarity™ Western ECL Substrate were obtained from Bio-Rad (Hercules, CA, USA). Protease inhibitor cocktail and lovastatin were purchased from Sigma (Saint Louis, MO, USA). Primary antibodies against Rab11A and Rap1A/Rap1B were obtained from Abcam (Cambridge, UK) and primary antibodies against β-actin along with secondary HRP-linked antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA).
Iodoacetamide and WaLP were purchased from Sigma (Saint Louis, MO, USA), DTT, CuSO4, TCEP from Fluorochem (Derbyshire, United Kingdom) and THPTA, biotin-PEG3-azide from Click Chemistry Tools (Scottsdale, AZ, USA). Sequencing grade chymotrypsin was purchased from Promega (Madison, WI, USA).
Iodoacetamide and WaLP were purchased from Sigma (Saint Louis, MO, USA), DTT, CuSO4, TCEP from Fluorochem (Derbyshire, United Kingdom) and THPTA, biotin-PEG3-azide from Click Chemistry Tools (Scottsdale, AZ, USA). Sequencing grade chymotrypsin was purchased from Promega (Madison, WI, USA).
9
Protease Activity Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Materials. Aeromonas proteolytica aminopeptidase (BLAP, PDB 1RTQ, Sigma, 116.51 units mg -1 ), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA, BioUltra, 99.5-101.5 %), iodoacetamide (IAM, 99 %), DL-dithio-threitol (DTT, 99 %), perinaphthenone (PN, 97 %), 7-hydroxy-4-methylcoumarin, 7-leucine-7-amido-4-methylcoumarin hydrochloride, were purchased from Sigma. Furfuryl alcohol (FFA, Merck, 98%) was distilled prior to use.
ProteaseMAX TM (Promega, lyophilized, V207A), sequencing grade chymotrypsin (Promega, lyophilized, V106A), iRT peptides (iRT Kit, Biognosys) were used for protein digestion and analytical measurements, respectively. Further chemicals purchased from commercial vendors are listed in Section S1.
ProteaseMAX TM (Promega, lyophilized, V207A), sequencing grade chymotrypsin (Promega, lyophilized, V106A), iRT peptides (iRT Kit, Biognosys) were used for protein digestion and analytical measurements, respectively. Further chemicals purchased from commercial vendors are listed in Section S1.
10
Protein Quantification and Trypsin Digestion
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!