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Protease C

Protease C is a critical enzyme involved in numerous biological processes.
It plays a key role in protein degradation and activation, making it an important target for pharmaceutical research.
PubCompare.ai's AI-driven tools help researchers optimize Protease C protocols by comparing the latest literature, preprints, and patents to identify the most accurate and reproducible methods.
Enhance your Protease C research with PubCompare.ai's powerful comparison features and improve the accuracy of your findings.

Most cited protocols related to «Protease C»

1
Plasmid Construction and Mutagenesis
Cited 375 times
Plasmid pDESTSIRV30, pDESTSIRV33 expressing the SIRV proteins (CAG38830 and CAG38833), pDESTAVRA expressing MRSA vraR protein (CAG40961) and pDESTFaBH2 expressing Pseudomonas aeruginosa FaBH2 protein (AAG06721)[28 (link)] were constructed using a modified Gateway technology with an N-terminal TEV protease cleavable His tag [29 (link)]. All the plasmids were propagated in DH5α E. coli cells (Stratagene, La Jolla) and plasmids were prepared using Qiagen miniprep kits (Qiagen, Germany). Pfu DNA polymerase, DpnI restriction enzyme are provided with QuikChange™ kit purchased from Stratagene, additional Pfu DNA polymerase was purchased from Promega when required. All the primers were synthesized by Eurogentec and simply purified by SePOP desalting. The melting temperature was calculated as Tm = 81.5 + 16.6(log([K+]/(1+0.7 [K+])) + 0.41(% [G+C]) – 500/(probe length in base) – 1.0(%mismatch) [30 (link)]. The Tm pp and Tm no were calculated for each primer. All primers and their Tm no and Tm pp are detailed in Table 1. PCR cycling was carried out using a Px2 thermal cycler (Thermo Electro Cooperation).
For single-site mutation, deletion or insertion, the PCR reaction of 50 μl contained 2–10 ng of template, 1 μM primer pair, 200 μM dNTPs and 3 units of Pfu DNA polymerase. The PCR cycles were initiated at 95°C for 5 minutes to denature the template DNA, followed by 12 amplification cycles. Each amplification cycle consisted of 95°C for 1 minute, Tm no -5°C for 1 minute and 72°C for 10 minutes or 15 minutes according to the length of the template constructs (about 500 bp per minute for Pfu DNA polymerase). The PCR cycles were finished with an annealing step at Tm pp-5 for 1 minute and an extension step at 72°C for 30 minutes. The PCR products were treated with 5 units of DpnI at 37°C for 2 hours and then 10 μl of each PCR reactions was analyzed by agarose gel electrophoresis. The full-length plasmid DNA was quantified by band density analysis against the 1636-bp band (equal to 10% of the mass applied to the gel) of the DNA ladders. An aliquot of 2 μl above PCR products, the PCR products generated using QuickChange™ or generated as described in [13 (link)] was transformed respectively into E. coli DH5α competent cells by heat shock. The transformed cells were spread on a Luria-Bertani (LB) plate containing antibiotics and incubated at 37°C over night. The number of colonies was counted and used as an indirect indication of PCR amplification efficiency. Four colonies from each plate were grown and the plasmid DNA was isolated. To verify the mutations, 500 ng of plasmid DNA was mixed with 50 pmole of T7 sequencing primer in a volume of 15 μl. DNA sequencing was carried out using the Sequencing Service, University of Dundee. For multiple site-directed mutations, deletions and insertions, the PCR was carried out in 50 μl of reaction containing 10 ng of template, 1 μM of each of the two primer pairs, 200 μM dNTPs and 3 units of Pfu DNA polymerase. The PCR cycles, DNA quantification, transformation and mutation verification were essentially the same as described above.
Liu H, & Naismith J.H. (2008). An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnology, 8, 91.
Publication 2008
Antibiotics Cells Deletion Mutation DNA Restriction Enzymes Electrophoresis, Agar Gel Escherichia coli Gene Deletion Heat-Shock Response Insertion Mutation Methicillin-Resistant Staphylococcus aureus Mutation Oligonucleotide Primers Pfu DNA polymerase Plasmids Promega Proteins Pseudomonas aeruginosa TEV protease
2
Automated Ligand Charge Calculation
Cited 288 times
The calculation of ligand charges necessitates detailed information on molecular structure and protonation states due to the large variation in the covalent structures of small-molecule protein ligands. The current version of PDB2PQR therefore requires the ligand structure, protonation state and formal charge to be specified by the user in the popular MOL2 (24 ) format. Ligand structures in MOL2 format are readily available from popular molecular modeling software and free web services such as PRODRG (25 (link)). Future versions of PDB2PQR will include a pdb2mol2 parser and automatic assignment of default ligand protonation states from a small-molecule pKa database.
The calculation of ligand charges in PDB2PQR is based on the partial equalization of orbital electronegativities (PEOE) procedure developed by Gasteiger and Marsili (26 ). In the PEOE procedure, orbital electronegativities χ are linked to partial atomic charges q by a polynomial expansion (χ = a +b·q + c·q2 + d·q3). The coefficients a, b, c and d were optimized by Gasteiger and Marsili using gas phase data on ionization potentials and electron affinities. We utilize a PEOE algorithm, which has been optimized by Czodrowski et al. to obtain better agreement between theoretical and experimental solvation energies for a set of small molecules including the polar amino acids (27 (link)). The resulting PEOE_PB charges have been tested for small-molecule complexes with trypsin, thrombin (28 (link)) and HIV protease (29 ), and have been found to give results that are in agreement with experimental values.
Dolinsky T.J., Czodrowski P., Li H., Nielsen J.E., Jensen J.H., Klebe G, & Baker N.A. (2007). PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucleic Acids Research, 35(Web Server issue), W522-W525.
Publication 2007
Amino Acids Electrons HIV Protease Ligands Molecular Structure Proteins Thrombin Trypsin
3
Nuclei Isolation for ATAC-Seq Analysis
Cited 196 times
See Supplementary
Protocol 2 for a detailed protocol. This protocol is highly similar
to the INTACT method19 (link) and
either protocol can be used for the isolation of nuclei with equivalent results.
All of the steps were carried out at 4 °C. A frozen tissue fragment ~20
mg was placed into a pre-chilled 2-ml Dounce homogenizer containing 2 ml of cold
1× homogenization buffer (320 mM sucrose, 0.1 mM EDTA, 0.1%
NP40, 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris pH 7.8,
1× protease inhibitors (Roche, cOmplete), and 167 μM
β-mercaptoethanol, in water). Tissue was homogenized with approximately
ten strokes with the loose ‘A’ pestle, followed by 20 strokes
with the tight ‘B’ pestle. Connective tissue and residual debris
were precleared by filtration through an 80-μm nylon mesh filter
followed by centrifugation for 1 min at 100 r.c.f. While avoiding the pelleted
debris, 400 μl was transferred to a pre-chilled 2-ml round bottom
Lo-Bind Eppendorf tube. An equal volume (400 μl) of a 50%
iodixanol solution (50% iodixanol in 1× homogenization buffer)
was added and mixed by pipetting to make a final concentration of 25%
iodixanol. 600 μl of a 29% iodixanol solution (29%
iodixanol in 1× homogenization buffer containing 480 mM sucrose) was
layered underneath the 25% iodixanol mixture. A clearly defined
interface should be visible. In a similar fashion, 600 μl of a
35% iodixanol solution (35% iodixanol in 1×
homogenization containing 480 mM sucrose) was layered underneath the 29%
iodixanol solution. Again, a clearly defined interface should be visible between
all three layers. In a swinging-bucket centrifuge, nuclei were centrifuged for
20 min at 3,000 r.c.f. After centrifugation, the nuclei were present at the
interface of the 29% and 35% iodixanol solutions. This band with
the nuclei was collected in a 300 μl volume and transferred to a
pre-chilled tube. Nuclei were counted after addition of trypan blue, which
stains all nuclei due to membrane permeabilization from freezing. 50,000 counted
nuclei were then transferred to a tube containing 1 ml of ATAC-seq RSB with
0.1% Tween-20. Nuclei were pelleted by centrifugation at 500 r.c.f. for
10 min in a pre-chilled (4 °C) fixed-angle centrifuge. Supernatant was
removed using the two pipetting steps described above. Because the nuclei were
already permeabilized, no lysis step was performed, and the transposition mix
(25 μl 2× TD buffer, 2.5 μl transposase (100 nM final),
16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl
10% Tween-20, 5 μl water) was added directly to the nuclear
pellet and mixed by pipetting up and down six times. Transposition reactions
were incubated at 37 °C for 30 min in a thermomixer with shaking at
1,000 r.p.m. Reactions were cleaned up with Zymo DNA Clean and Concentrator 5
columns. The remainder of the ATAC-seq library preparation was performed as
described previously18 .
Corces M.R., Trevino A.E., Hamilton E.G., Greenside P.G., Sinnott-Armstrong N.A., Vesuna S., Satpathy A.T., Rubin A.J., Montine K.S., Wu B., Kathiria A., Cho S.W., Mumbach M.R., Carter A.C., Kasowski M., Orloff L.A., Risca V.I., Kundaje A., Khavari P.A., Montine T.J., Greenleaf W.J, & Chang H.Y. (2017). An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nature methods, 14(10), 959-962.
Publication 2017
2-Mercaptoethanol ATAC-Seq Buffers Cell Nucleus Centrifugation Cerebrovascular Accident Connective Tissue Digitonin DNA Library Edetic Acid Filtration iodixanol isolation Nylons Protease Inhibitors Sucrose Tissue, Membrane Tissues Transposase Tromethamine Trypan Blue Tween 20
4
Polysome Immunoprecipitation and RNA Extraction
Cited 123 times

Protocol full text hidden due to copyright restrictions

Open the protocol to access the free full text link

Publication 2008
1,2-dihexadecyl-sn-glycero-3-phosphocholine Alabaster austin Brain Stem Buffers Cells Cerebellum Chloroform Cholinergic Agents Cold Temperature Cycloheximide Deoxyribonucleases Digestion Dithiothreitol Endoribonucleases Ethanol G-substrate Goat HEPES inhibitors Isopropyl Alcohol Lipids Magnesium Chloride Mice, Laboratory Mice, Transgenic Motor Neurons Nonidet P-40 Polyribosomes Protease Inhibitors Purkinje Cells Ribosomal RNA RNA, Messenger Sodium Acetate Sodium Chloride Striatum, Corpus Teflon Tissues trizol
5
Biotinylated Protein Isolation Protocol
Cited 101 times
Cells were incubated for 24 h in complete media supplemented with 1 µg/ml doxycycline and 50 µM biotin. After three PBS washes, cells (for small-scale analysis, <107; for large scale analysis, 4 × 107) were lysed at 25°C in 1 ml lysis buffer (50 mM Tris, pH 7.4, 500 mM NaCl, 0.4% SDS, 5 mM EDTA, 1 mM DTT, and 1x Complete protease inhibitor [Roche]) and sonicated. Triton X-100 was added to 2% final concentration. After further sonication, an equal volume of 4°C 50 mM Tris (pH 7.4) was added before additional sonication (subsequent steps at 4°C) and centrifugation at 16,000 relative centrifugal force. Supernatants were incubated with 600 µl Dynabeads (MyOne Steptavadin C1; Invitrogen) overnight. Beads were collected and washed twice for 8 min at 25°C (all subsequent steps at 25°C) in 1 ml wash buffer 1 (2% SDS in dH2O). This was repeated once with wash buffer 2 (0.1% deoxycholate, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, and 50 mM Hepes, pH 7.5), once with wash buffer 3 (250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, and 10 mM Tris, pH 8.1) and twice with wash buffer 4 (50 mM Tris, pH 7.4, and 50 mM NaCl). 10% of the sample was reserved for Western blot analysis. Bound proteins were removed from the magnetic beads with 50 µl of Laemmli SDS-sample buffer saturated with biotin at 98°C. For the larger scale preparation, 90% of the sample to be analyzed by mass spectrometry was washed twice in 50 mM NH4HCO3.
Roux K.J., Kim D.I., Raida M, & Burke B. (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. The Journal of Cell Biology, 196(6), 801-810.
Publication 2012
Biotin Buffers Cells Centrifugation Deoxycholate Doxycycline Edetic Acid HEPES Laemmli buffer Mass Spectrometry Nonidet P-40 Protease Inhibitors Proteins Sodium Chloride Triton X-100 Tromethamine Western Blot

Most recents protocols related to «Protease C»

1
Immobilization of Glu-C Enzyme on PDA/PEI/AM-KIT-6
Not available on PMC !
To prevent cross-enzymolysis, a stepwise fixation method was employed for the immobilization of Glu-C on PDA/PEI/AM-KIT-6, based on previous work with some modifications [39] (link). Typically, PDA/PEI/AM-KIT-6 was dispersed in 2 mL of phosphate buffer (PBS, 50 mM, pH 6.0), followed by the addition of 1.0 mg of Glu-C and 5 μL of a 20 wt % aqueous solution of GA. After stirring for 24 hours at 30℃, the solids were collected by centrifugation (10,000 rpm) for 10 minutes and washed several times with PBS to remove residual reactants. The resulting immobilized Glu-C was labeled as PDA/PEI/AM-KIT-6-Glu-C.
Yuan F., Lü X., Han X., Qin T., Wang P., & Bai H. (2024). Rapid digestion of proteins with a dual-enzyme microreactor featuring 3-D pores formed by dopamine/polyethyleneimine/ acrylamide-coated KIT-6 molecular sieve.
Publication 2024
2
Protease Assay Protocols for SARS-CoV-2, MMP9, and TEV
SARS-CoV-2 Mpro: The assay was performed with a buffer consisting of 50 mM Tris-HCl and 1 mM EDTA (pH 7.3) at 30°C for various lengths of time. 1 μg of Mpro (Merck Darmstadt, Germany) of 33.8 kDa in size with 50 µg of protease sensor 33.1 kDa.
MMP9: The assay was performed with a buffer consisting of 50 mm Tris/HCl, 150 mm NaCl, 10 mm CaCl2, 20 µM ZnCl2, 0.05% Brij35 at pH = 7.5.1 μg of MMP9 catalytic domain (Abcam, Cambridge, UK) of 40 kDa combined with 50 µg of protease sensor 33.1 kDa for various lengths of time at 37°C.
TEV: Carried out as per manufacturer’s instructions (NEB, Ipswich, MA, USA). 1 μL of TEV Protease per 15 μg of substrate in the provided TEV reaction buffer and incubated at 30°C for various lengths of time.
Devoy C., Flores Bueso Y., Buckley S., Walker S, & Tangney M. (2024). Synthetic protein protease sensor platform. Frontiers in Bioengineering and Biotechnology, 12, 1347953.
Publication 2024
3
Structural Analysis of HIV Protease Subtype C
The structures for the closed conformation of wild-type and L38HL of HIV protease subtype C are obtained through homology modeling using MODELLER (Webb and Sali, 2016 ) based on the structure of HIV protease subtype B in closed conformation as template (PDB code: 2AQU). The modeled structures were then docked with the ATV using AutoDock (Forli et al., 2016 (link)). The conformation with the lowest binding energy was chosen for further analysis. The docked structures were then used as the initial co-ordinates for MD simulations.
Sankaran S.V., Krishnan S.R., Sayed Y, & Gromiha M.M. (2024). Mechanism of drug resistance in HIV-1 protease subtype C in the presence of Atazanavir. Current Research in Structural Biology, 7, 100132.
Publication 2024
4
Protease Activity Inhibition Assay
Not available on PMC !

Protocol full text hidden due to copyright restrictions

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Publication 2024
5
High-throughput screening of ZIKV protease
Not available on PMC !

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Publication 2024

Top products related to «Protease C»

1
Protease inhibitor cocktail by Roche
Sourced in United States, Switzerland, Germany, China, United Kingdom, France, Canada, Japan, Italy, Australia, Austria, Sweden, Spain, Cameroon, India, Macao, Belgium, Israel
Protease inhibitor cocktail is a laboratory reagent used to inhibit the activity of proteases, which are enzymes that break down proteins. It is commonly used in protein extraction and purification procedures to prevent protein degradation.
2
Protease inhibitor cocktail by Merck Group
Sourced in United States, Germany, China, United Kingdom, Italy, Japan, Sao Tome and Principe, France, Canada, Macao, Switzerland, Spain, Australia, Israel, Hungary, Ireland, Denmark, Brazil, Poland, India, Mexico, Senegal, Netherlands, Singapore
The Protease Inhibitor Cocktail is a laboratory product designed to inhibit the activity of proteases, which are enzymes that can degrade proteins. It is a combination of various chemical compounds that work to prevent the breakdown of proteins in biological samples, allowing for more accurate analysis and preservation of protein integrity.
3
Pvdf membrane by Merck Group
Sourced in United States, Germany, China, United Kingdom, Morocco, Ireland, France, Italy, Japan, Canada, Spain, Switzerland, New Zealand, India, Hong Kong, Sao Tome and Principe, Sweden, Netherlands, Australia, Belgium, Austria
PVDF membranes are a type of laboratory equipment used for a variety of applications. They are made from polyvinylidene fluoride (PVDF), a durable and chemically resistant material. PVDF membranes are known for their high mechanical strength, thermal stability, and resistance to a wide range of chemicals. They are commonly used in various filtration, separation, and analysis processes in scientific and research settings.
4
Protease inhibitor by Roche
Sourced in United States, Switzerland, Germany, China, United Kingdom, Canada, France, Japan, Italy, Australia, Spain, India, Macao, Netherlands, Denmark, Belgium, Cameroon
Protease inhibitors are a class of laboratory equipment used in the field of biochemistry and molecular biology. These inhibitors are designed to specifically target and inactivate proteases, which are enzymes that break down proteins. Protease inhibitors play a crucial role in various experimental and analytical procedures, such as protein extraction, purification, and stabilization.
5
Complete protease inhibitor cocktail by Roche
Sourced in United States, Germany, Switzerland, United Kingdom, China, France, Japan, Canada, Spain, Belgium, Australia, Sweden, Italy, Ireland, Macao
The Complete Protease Inhibitor Cocktail is a laboratory product designed to inhibit a broad spectrum of proteases. It is a concentrated solution containing a mixture of protease inhibitors effective against a variety of protease classes. This product is intended to be used in research applications to preserve the integrity of target proteins by preventing their degradation by proteolytic enzymes.
6
Pierce bca protein assay kit by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, China, Australia, Switzerland, France, Italy, Canada, Spain, Japan, Belgium, Sweden, Lithuania, Austria, Denmark, Poland, Ireland, Portugal, Finland, Czechia, Norway, Macao, India, Singapore
The Pierce BCA Protein Assay Kit is a colorimetric-based method for the quantification of total protein in a sample. It utilizes the bicinchoninic acid (BCA) reaction, where proteins reduce Cu2+ to Cu+ in an alkaline environment, and the resulting purple-colored reaction is measured spectrophotometrically.
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
Protease inhibitor by Merck Group
Sourced in United States, Germany, China, United Kingdom, Italy, Macao, Sao Tome and Principe, Canada, Japan, France, Switzerland, Israel
Protease inhibitors are a class of pharmaceutical compounds that work by inhibiting the activity of proteases, which are enzymes that break down proteins. They are commonly used in the treatment of various conditions, including viral infections and certain types of cancer. Protease inhibitors function by binding to and blocking the active site of proteases, preventing them from carrying out their enzymatic activities.
9
Ripa buffer by Merck Group
Sourced in United States, Germany, United Kingdom, Switzerland, Italy, Sao Tome and Principe, China, Canada, Macao, France, Japan, Spain, Ireland, Australia, Sweden, Belgium, Denmark, Morocco, Austria, Poland
RIPA buffer is a widely used lysis buffer for extracting proteins from cells and tissues. It is a detergent-based buffer that helps solubilize proteins, disrupt cell membranes, and maintain the integrity of protein structures during the extraction process. The buffer contains a combination of ionic and non-ionic detergents, as well as other components that help stabilize and preserve the extracted proteins.
10
Ripa buffer by Thermo Fisher Scientific
Sourced in United States, Germany, China, Switzerland, Japan, United Kingdom, Italy, Australia, Sweden, Belgium, Canada, Denmark, France, Sao Tome and Principe, Macao, Austria, Morocco, Norway, Israel
RIPA buffer is a commonly used lysis buffer for the extraction and solubilization of proteins from cells and tissues. It contains a mixture of ionic and non-ionic detergents that help disrupt cell membranes and release cellular contents, including proteins. The buffer also includes salts and buffers to maintain the pH and ionic strength of the solution.

More about "Protease C"

Protease C, also known as Peptidase C or Endopeptidase C, is a critical enzyme involved in numerous biological processes.
It plays a key role in protein degradation and activation, making it an important target for pharmaceutical research.
This enzyme is responsible for cleaving peptide bonds within proteins, which is essential for processes like cell signaling, immune response, and tissue remodeling.
Researchers often use Protease inhibitor cocktails, which contain a mixture of compounds that block the activity of various proteases, including Protease C.
PVDF membranes are also commonly used in proteomic studies to capture and analyze Protease C and other enzymes.
Complete protease inhibitor cocktails, which include inhibitors for a wide range of proteases, can be used to ensure comprehensive protection of proteins during sample preparation and analysis.
The Pierce BCA Protein Assay Kit, also known as the BCA protein assay kit, is a widely used method for quantifying total protein levels, including Protease C, in biological samples.
RIPA buffer is another important tool for researchers studying Protease C, as it helps to solubilize and extract proteins from cells and tissues.
PubCompare.ai's AI-driven tools can help researchers optimize their Protease C protocols by comparing the latest literature, preprints, and patents to identify the most accurate and reproducible methods.
This can enhance the accuracy and reliability of your Protease C research findings.