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Chymostatin

Chymostatin is a potent and selective inhibitor of chymotrypsin-like serine proteases, such as chymotrypsin, cathepsin G, and elastase.
It is commonly used in biological research to investigate the role of these enzymes in various physiological and pathological processes.
Chymostatin has been studied for its potential therapeutic applications, particularly in the areas of inflammation, cancer, and neurodegenerative diseases.
Researchers can leverage PubCompare.ai's AI-driven platform to easily locate protocols from literature, pre-prints, and patents related to chymostatin research, and use the tool's powerful comparision features to identify the best protocols and products for their specific needs, optimizing their chymostatin-based studies.

Most cited protocols related to «Chymostatin»

1
Protein Extraction and Immunoblot Analysis
Cited 44 times
Total proteins were extracted from 100 mg of sample using extraction buffer (100 mM Tris-Cl pH8, 150 mM NaCl, 0.6% IGEPAL, 1 mM EDTA, 3 mM DTT with protease inhibitors, PMSF, leupeptin, aprotinin, pepstatin, antipain, chymostatin, Na2VO3, NaF, MG132, and MG115. Proteins were separated on a 10% polyacrylamide gel. Immunoblot analysis was carried out using mouse α-GFP (1:2000; Invitrogen) for TuMV GFP and rat α-HA (1:500) antibody for pCas13a. The antigens were detected by chemiluminescence using an ECL-detecting reagent (Thermo Scientific).
Aman R., Ali Z., Butt H., Mahas A., Aljedaani F., Khan M.Z., Ding S, & Mahfouz M. (2018). RNA virus interference via CRISPR/Cas13a system in plants. Genome Biology, 19, 1.
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Publication 2018
Antigens Antipain Aprotinin Buffers Chemiluminescence chymostatin Edetic Acid Elafin Immunoblotting Immunoglobulins leupeptin MG 115 MG 132 Mice, House pepstatin polyacrylamide gels Proteins Sodium Chloride Tromethamine
2
Purification of Ndc80 Complex from S. cerevisiae and Humans
Cited 11 times

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Publication 2009
Aprotinin Benzonase Buffers Chromatography, Affinity chymostatin Claw Cloning Vectors Gel Chromatography Genes HEPES his6 tag Homo sapiens leupeptin NDC80 protein, human pepstatin Phenylmethylsulfonyl Fluoride Protease Inhibitors Protein Subunits Resins, Plant Saccharomyces cerevisiae Sodium Chloride Strains
3
Western Blot Analysis of Protein Expression
Cited 10 times
Cells were washed twice with ice-cold PBS, and lysed in 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM PMSF, 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/mL chymostatin). Protein concentration was determined using Bradford reagent (Bio-Rad, Hercules, CA). Equal amounts of lysate (20 μg) were resolved by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). Thereafter, the membrane was blocked with 1 × TBS containing 0.05% Tween 20 (TBST) and 5% skim milk or 2% BSA for 1 h at room temperature. After blocking, the membranes were incubated overnight at 4 °C with the respective primary antibodies, washed with 1 × TBST, and then incubated with diluted horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000, Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. After three washes, the bound antibodies were detected using an enhanced chemiluminescence (ECL) kit (Millipore, Bedford, MA).
Park K.R., Kim E.C., Hong J.T, & Yun H.M. (2018). Dysregulation of 5-hydroxytryptamine 6 receptor accelerates maturation of bone-resorbing osteoclasts and induces bone loss. Theranostics, 8(11), 3087-3098.
Publication 2018
Antibodies Aprotinin Cells Chemiluminescence chymostatin Cold Temperature Horseradish Peroxidase Milk, Cow's pepstatin Polyacrylamide Gel Electrophoresis polyvinylidene fluoride Protease Inhibitors Proteins Sodium Tissue, Membrane Tromethamine Tween 20
4
Purification and Fractionation of Chromaffin Secretory Vesicles
Cited 9 times
Dense core secretory vesicles, represented by chromaffin secretory vesicles (also known as chromaffin granules), were purified from fresh bovine adrenal medulla by differential sucrose density gradient centrifugation, as described previously (37 ,38 (link)), involving extensive wash steps to obtain purified chromaffin granules. We have documented the high purity of this preparation of isolated secretory vesicles by electron microscopy (Fig. 1) and biochemical markers (36 (link)–38 (link)). Sucrose gradient purification results in a preparation of purified, intact chromaffin secretory vesicles that lack biochemical markers for the subcellular organelles of lysosomes (acid phosphatase marker) (38 (link)), cytoplasm (lactate dehydrogenase marker) (37 ), mitochondria (fumarase and glutamate dehydrogenase markers) (36 (link),37 ), and endoplasmic reticulum (glucose-6-phosphatase marker) (37 ). Enzyme markers have been measured in the purified chromaffin secretory vesicle preparation as 1% or less of total homogenate markers, which, thus, indicate the high purity of these isolated secretory vesicles (36 (link)–38 (link)).
In addition, this study further assessed the removal of the lysosomal enzyme marker acid phosphatase from the purified preparation of chromaffin granules compared to unpurified sample of chromaffin granules obtained at an early step in the purification procedure (illustrated in figure 1a). Purified and unpurified granules were analyzed on a multi-step sucrose gradient of 2.2 to 1.2 M sucrose (2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, and 1.2 M sucrose steps each consisting of 2.5 ml) by ultracentrifugation at 120,000 × g in a SW28 rotor (25,000 rpm) at 4° C for 100 min. Gradient fractions of 0.5 ml were collected from the bottom of the tube (2.2 M sucrose), and fractions were assayed for (Met)enkephalin by RIA as previously described (5 (link)) as a marker for chromaffin granules, and acid phosphatase activity as a marker for lysosomes as described previously (38 (link)). Results show that the purified chromaffin granules lack acid phosphatase activity, indicating effective removal of lysosomes of density near that of chromaffin granules (explained in fig. 1 of results). These new data and established purity in the literature (36 (link)–38 (link)) document the purity of these chromaffin secretory vesicles for this study.
Soluble and membrane components of the purified chromaffin granules were prepared by lysing (by freeze-thawing) purified chromaffin granules in isotonic buffer conditions consisting of 150 mM NaCl in 50 mM Na-acetate, pH 6.0, with a cocktail of protease inhibitors (10 μM pepstatin A, 10 μM leupeptin, 10 μM chymostatin, 10 μM E64c, and 1 mM AEBSF). The lysed granules were centrifuged at 100,000 × g (SW60 rotor) at 4° C for 30 minutes. The resultant supernatant was collected as the soluble fraction. The pellet was collected as the membrane fraction, and washed two times by re-suspending in the lysis buffer and centrifugation (100,000 × g, 30 min). The final pellet was resuspended in the lysis buffer and designated as the membrane fraction.
The soluble and membrane fractions were each subjected to removal of the abundant chromogranin A (CgA) protein, by its binding to calmodulin-Sepharose (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ) (39 (link)). The soluble fraction and membrane fraction (solubilized in 50 mM CHAPS) were each incubated with a slurry of calmodulin-Sepharose at 4° C overnight in equilibration buffer (50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 2 mM CaCl2, and protease inhibitors consisting of 5 μM E64c, 5 μM leupeptin, 5 μM chymostatin, 5 μM pepstatin A, 5 μM bestatin, 1 μM GEMSA, and 50 mM PMSF). The mixture was centrifuged and the supernatant collected as the soluble fraction without CgA. This step removed approximately 90–95% of CgA, based on assessment by anti-CgA western blots.
Proteins in the membrane fraction were concentrated by chloroform-methanol precipitation. To the membrane fraction (400 μg in 300 μl) was added MeOH (400 μl), chloroform (100 μl), and deionized water (300 μl) with mixing between each step, followed by centrifugation (14,000 × g for 1 min). The top aqueous layer was removed, while retaining the protein precipitate at the top of the chloroform layer; after addition of MeOH (400 μl), mixing, and centrifugation (14,000 × g for 2 min), the pelleted protein was collected for trypsin digestion (40 (link)).
Wegrzyn J.L., Bark S.J., Funkelstein L., Mosier C., Yap A., Kazemi-Esfarjani P., La Spada A., Sigurdson C., O’Connor D.T, & Hook V. (2010). Proteomics of Dense Core Secretory Vesicles Reveal Distinct Protein Categories for Secretion of Neuroeffectors for Cell-Cell Communication. Journal of proteome research, 9(10), 5002-5024.
Publication 2010
5
Xenopus Embryo Cytoplasmic Extract Preparation
Cited 8 times

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Publication 2010
Adenosine Triphosphate Cell Nucleus chymostatin Cycloheximide Cytochalasin D Cytoplasm Egtazic Acid Embryo HEPES Interphase leupeptin Magnesium Chloride pepstatin Phosphocreatine Sucrose Xenopus laevis

Most recents protocols related to «Chymostatin»

1
Inhibition of Norovirus Protease by Novel Compounds
Not available on PMC !

Example 12

The ability of these compounds to inhibit the NoV, specifically Minerva virus protease catalytic Cys139 covalently (IC50 and Ki) was determined with an enzyme kinetic assay. NoV strains, specifically GII.4 such as the Minerva virus are responsible for causing the majority (˜80%) of infections in humans. The activity of the inhibitors was evaluated by monitoring the cleavage of a FRET substrate every one minute for 20 minutes (excitation/emission: 488/520 nm) using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale CA). Serial dilutions of each inhibitor were incubated with enzyme for 90 minutes at 37° C. before addition of the FRET substrate to ensure complete inactivation. Commercially available protease inhibitors chymostatin and rupintrivir were used as controls.

TABLE 4
CompoundIC50 (μM)Ki (μM)
110.112 ± 0.0250.427 ± 0.109
190.150 ± 0.002 1.19 ± 0.444
230.204 ± 0.009 1.59 ± 0.050
290.140 ± 0.0170.670 ± 0.019
350.167 ± 0.0050.858 ± 0.032
36 1.17 ± 0.333 3.60 ± 0.501
372.63 ± 1.0414.03 ± 5.55 
38>10ND
39>10ND
64>100ND
6730.0 ± 1.8 >100
830.482 ± 0.07 7.095 ± 5.583
84>100ND
chymostatin13.71.6 ± 1.0
rupintrivir23.68.2 ± 2.3

US11859014B2. Peptidomimetics for the treatment of Norovirus infection (2024-01-02). EMORY UNIVERSITY [US]. Inventors: Raymond F Schinazi [US], Franck Amblard [US], Ladislau Kovari [US], Peng Liu [US], Shaoman Zhou [US], Benjamin D Kuiper [US], BRadley J Keusch [US].
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Patent 2024
Cardiac Arrest Catalysis chymostatin Cytokinesis Enzyme Assays Enzymes Fluorescence Resonance Energy Transfer Homo sapiens Infection inhibitors Kinetics Medical Devices Norovirus Infection Peptide Hydrolases Peptidomimetics Protease Inhibitors rupintrivir Strains Technique, Dilution Virus
2
Tissue Homogenization and Enzyme Extraction
Tissue samples (approximately 0.25 g) were homogenized with 2 mL of 100 mmol/L Tris buffer (pH 8.0) containing 2 mmol/L EDTA, 3 mmol/L dithiothreitol (DTT), 0.5% Triton X-100, and 0.1% protease inhibitors (aprotinin, chymostatin, pepstatin A, and phenylmethylsulfonylfluoride, 2.5 μg/mL each in dimethyl sulfoxide). The homogenates were centrifuged at 10,000 × g for 1 min at 25 °C. The supernatant fluid (enzyme extract) was used for assays of enzymatic activities.
He W., Li X, & Wu G. (2024). Dietary glycine supplementation enhances syntheses of creatine and glutathione by tissues of hybrid striped bass (Morone saxatilis ♀ × Morone chrysops ♂) fed soybean meal-based diets. Journal of Animal Science and Biotechnology, 15, 67.
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Publication 2024
3
Evaluation of MdpS Protease Inhibitors
Potential inhibition of the proteolytic activity of MdpS was studied with a panel of protease inhibitors: AEBSF, ALLN, antipain, bestatin, chymostatin, E64, leupeptin, pepstatin, phosphoramidon, and PMSF (G-Biosciences, Geno Technology Inc., United States) following the manufacturer’s instruction. EDTA-Na2 was excluded from the panel since its cation-chelating effect was shown in the physicochemical tests described above. MdpS inhibition was analyzed by gel electrophoresis using Etanercept as the substrate, followed by densitometric quantification.
Leo F., Lood R., Thomsson K.A., Nilsson J., Svensäter G, & Wickström C. (2024). Characterization of MdpS: an in-depth analysis of a MUC5B-degrading protease from Streptococcus oralis. Frontiers in Microbiology, 15, 1340109.
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Publication 2024
4
Chymotrypsin Inhibitory Activity Assay
Not available on PMC !
The inhibitory activity of chymotrypsin was carried out according to the method described by Saleem et al. [26] (link). A mixture containing 60 μl of 50 mM Tris-HCl buffer (pH 7.6), 15 μl (0.9 units) of chymotrypsin (Sigma, USA) and varying concentrations (15-240 µg/mL) of betulinic acid or the standard protease inhibitor, chymostatin was incubated at 37°C for 20 min. After incubation, 15 μL of N-succinyl phenyl-alanine-p-nitroanilide (1.3 mM) was added to the mixture and incubated for another 30 min (37°C). The absorbance was read at 410 nm, and the percentage inhibition was calculated as
Salau V.F., Erukainure O.L., Aljoundi A., Akintemi E.O., Elamin G, & Odewole O.A. (2024). Exploring the inhibitory action of betulinic acid on key digestive enzymes linked to diabetes via in vitro and computational models: approaches to anti-diabetic mechanisms. SAR and QSAR in environmental research, 35(5).
Publication 2024
5
Nucleosome Reconstitution and H1.8-GFP Binding
The 193 bp 601 DNA fragment was amplified by a PCR reaction (62 (link), 63 (link)). The nucleosomes were assembled with the salt dialysis method described above. The reconstituted nucleosome was dialyzed into buffer XL (80 mM PIPES-KOH [pH 6.8], 15 mM NaCl, 60 mM KCl, 30 % glycerol, 1 mM EGTA, 1 mM MgCl2, 10 mM β-glycerophosphate, 10 mM sodium butyrate). H1.8-GFP was mixed with nucleosome with a 1.25 molar ratio in the presence of 0.001 % poly L-glutamic acid (wt 3,000–15,000) (Sigma-Aldrich) and incubated at 37 °C for 30 min. As a control nucleosome sample without H1.8-GFP, the sample without H1.8-GFP was also prepared. The samples were then crosslinked adding a 0.5-time volume of buffer XL containing 3 % formaldehyde and incubating for 90 min on ice. The crosslink reaction was quenched by adding 1.7 volume of quench buffer (30 mM HEPES-KOH (pH 7.4), 150 mM KCl, 1 mM EGTA, 10 ng/μL leupeptin, 10 ng/μL pepstatin, 10 ng/μL chymostatin, 10 mM sodium butyrate, 10 mM β-glycerophosphate, 400 mM glycine, 1 mM MgCl2, 5 mM DTT). The quenched sample was layered onto the 10–25 % linear sucrose gradient solution with buffer SG (15 mM HEPES-KOH [pH 7.4], 50 mM KCl, 10–22 % sucrose, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml chymostatin, 10 mM sodium butyrate, 10 mM β-glycerophosphate, 1 mM EGTA, 20 mM glycine) and spun at 32,000 rpm (max 124,436 rcf) and 4 °C for 13 h using SW55Ti rotor in Optima L80 (Beckman Coulter). The centrifuged samples were fractionated from the top of the sucrose gradient. The concertation of H1.8-GFP bound nucleosome in each fraction is calculated based on the 260 nm light absorbance detected by Nanodrop 2000 (Thermo Scientific).
Arimura Y., Konishi H.A, & Funabiki H. (2024). MagIC-Cryo-EM: Structural determination on magnetic beads for scarce macromolecules in heterogeneous samples. bioRxiv.
Publication Preprint 2024

Top products related to «Chymostatin»

1
Chymostatin by Merck Group
Sourced in United States, Switzerland
Chymostatin is a protease inhibitor that functions by inhibiting chymotrypsin, a digestive enzyme found in the pancreas. It is a type of lab equipment used in biochemical research and analysis.
2
Leupeptin by Merck Group
Sourced in United States, Germany, Italy, China, Japan, France, Israel, Switzerland, Canada, Sao Tome and Principe, United Kingdom, Australia, Ireland
Leupeptin is a protease inhibitor that can be used in laboratory settings to inhibit the activity of certain proteases. It is a tripeptide compound that binds to and inhibits the catalytic sites of proteases.
3
Pepstatin by Merck Group
Sourced in United States, Germany, China, Switzerland, Canada, Japan
Pepstatin is a protease inhibitor that can selectively inhibit aspartic proteases, such as pepsin, renin, and cathepsin D. It is a naturally occurring peptide compound isolated from various Actinomycetes bacterial strains. Pepstatin functions by binding to the active site of aspartic proteases, thereby preventing their enzymatic activity.
4
Antipain by Merck Group
Sourced in United States, Germany, France
Antipain is a protease inhibitor used in laboratory settings. It functions by inhibiting the activity of certain enzymes, specifically serine and cysteine proteases. Antipain is commonly utilized in research applications to prevent unwanted proteolysis or protein degradation.
5
Bradford assay by Bio-Rad
Sourced in United States, Germany, United Kingdom, Italy, Canada, China, France, Switzerland, Austria, Spain, Japan, Australia, Brazil, Ireland, Sweden
The Bradford assay is a colorimetric protein assay used to measure the concentration of protein in a solution. It is based on the color change of the Coomassie Brilliant Blue G-250 dye in response to various concentrations of protein.
6
Aprotinin by Merck Group
Sourced in United States, Germany, United Kingdom, Italy, China, Canada, Japan, Spain, France, Israel, Belgium, Austria, Switzerland, Finland, India, Australia, Macao, Hungary, Sweden, Sao Tome and Principe
Aprotinin is a protease inhibitor derived from bovine lung tissue. It is used as a laboratory reagent to inhibit protease activity in various experimental procedures.
7
Bca protein assay kit by Thermo Fisher Scientific
Sourced in United States, China, Germany, United Kingdom, Japan, Belgium, France, Switzerland, Italy, Canada, Australia, Sweden, Spain, Israel, Lithuania, Netherlands, Denmark, Finland, India, Singapore
The BCA Protein Assay Kit is a colorimetric detection and quantification method for total protein concentration. It utilizes bicinchoninic acid (BCA) for the colorimetric detection and quantification of total protein. The assay is based on the reduction of Cu2+ to Cu1+ by protein in an alkaline medium, with the chelation of BCA with the Cu1+ ion resulting in a purple-colored reaction product that exhibits a strong absorbance at 562 nm, which is proportional to the amount of protein present in the sample.
8
Lowry protein assay kit by Merck Group
Sourced in United States
The Lowry protein assay kit is a colorimetric assay used to quantify the total protein concentration in a sample. It is based on the Lowry method, which involves the reaction of proteins with copper in an alkaline solution and the subsequent reduction of the Folin-Ciocalteu reagent. The resulting colored complex can be measured spectrophotometrically to determine the protein concentration in the sample.
9
Phenyl methyl sulfonyl fluoride (pmsf) by Merck Group
Sourced in United States, China, Germany, Switzerland, Italy, United Kingdom, Macao, Sao Tome and Principe, Japan, Canada, Spain, Poland, India, Australia
PMSF is a protease inhibitor used in biochemical research and laboratory applications. It functions by irreversibly inhibiting serine proteases, which are a class of enzymes involved in various biological processes. PMSF is commonly utilized in protein extraction and purification protocols to prevent proteolytic degradation of target proteins.
10
Hybond enhanced chemiluminescence ecl nitrocellulose membrane by Bio-Rad
Sourced in United States
Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane is a high-performance nitrocellulose membrane designed for western blotting applications. It is optimized for use with chemiluminescent detection systems, providing enhanced sensitivity and signal-to-noise ratio.

More about "Chymostatin"

Chymostatin is a potent and selective inhibitor of chymotrypsin-like serine proteases, such as chymotrypsin, cathepsin G, and elastase.
It is commonly used in biological research to investigate the role of these enzymes in various physiological and pathological processes.
Chymostatin has been studied for its potential therapeutic applications, particularly in the areas of inflammation, cancer, and neurodegenerative diseases.
Researchers can leverage PubCompare.ai's AI-driven platform to easily locate protocols from literature, pre-prints, and patents related to chymostatin research, and use the tool's powerful comparison features to identify the best protocols and products for their specific needs, optimizing their chymostatin-based studies.
Chymostatin is often used in conjunction with other protease inhibitors, such as leupeptin, pepstatin, and antipain, to create a comprehensive protease inhibitor cocktail.
These inhibitors target different classes of proteases, ensuring a broader spectrum of enzyme inhibition.
The Bradford assay and the BCA protein assay kit are commonly used methods for quantifying protein concentration, which is an important step in many chymostatin-based experiments.
The Lowry protein assay kit is another widely used alternative.
PMSF (phenylmethylsulfonyl fluoride) is another protease inhibitor that is often used in conjunction with chymostatin to prevent protein degradation during sample preparation and analysis.
The Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane is a popular choice for Western blotting, a technique that can be used to detect and quantify specific proteins, including those affected by chymostatin treatment.
By leveraging the insights and tools provided by PubCompare.ai, researchers can optimize their chymostatin-based studies, leading to more reproducible and accurate results that advance our understanding of the role of chymotrypsin-like proteases in various biological processes and disease states.