Recombinant SARS-CoV-2 3CLpro (100 nM) was assayed at 25°C in either (a) in 30 μL reaction volumes containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 5 % glycerol, 0.01% Tween 20 100 μM of Mu-HSSKLQ-AMC (Sigma-Aldrich, SCP0224), in 384-well black plates in triplicate or (b) in 50 μL reaction mixtures containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 10% DMSO, and 25–50 nM 3CLpro in 96-well plates (Greiner, flat-bottom half volume, clear black plates), using the FRET-based substrate Abz-SAVLQSGFRK(DNP)-NH2 wherein peptidolysis was measured at 320/420 nm (ex/em) (Biotek® Synergy M2) in the presence and absence of 0–50 μM K777 in duplicate. Recombinant SARS-CoV-2 PL was assayed in either (a) 50 mM HEPES, 150 mM NaCl, 1 mM DTT, 0.01% Tween 20; pH 6.5 buffer using 50 μM Z-RLRGG-AMC (Bachem, I1690) and 24.5 nM enzyme or (b) 50 mM HEPES pH 7.5, 5 mM DTT, 0.1 mg/mL BSA, 2% DMSO, 50 μM Z-RLRGG-AMC, and 10 nM PL, where rates in the presence and absence of K777 were measured at 360/460 nm (ex/em). In some studies, enzymes were pre-incubated with 20 μM of K777 or 0.0025 % DMSO for 15 minutes and then diluted in an equal volume of substrate in assay buffer.
Z rlrgg amc
Manufactured by Bachem
Sourced in Switzerland
Z-RLRGG-AMC is a synthetic peptide used as a fluorogenic substrate for the measurement of protease activity. The product consists of the peptide sequence Z-Arg-Leu-Arg-Gly-Gly-AMC, where the 7-amino-4-methylcoumarin (AMC) moiety serves as a fluorescent reporter group.
Automatically generated - may contain errors
Lab products found in correlation
Magellan 7, by Tecan (2 mentions)
Nedd8 amc, by Enzo Life Sciences (2 mentions)
Infinite f200 pro plate, by Tecan (2 mentions)
Mu hssklq amc, by Merck Group (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
Mlv votu, by GenScript (1 mentions)
Infinite f200 pro plate reader, by Tecan (1 mentions)
Isg15 amc, by R&D Systems (1 mentions)
Sumo 2 amc, by R&D Systems (1 mentions)
Sumo1 amc, by R&D Systems (1 mentions)
7 protocols using z rlrgg amc
1
SARS-CoV-2 3CLpro and PL Inhibition Assays
2
Purification and Characterization of SARS-CoV and HCoV-NL63 Proteases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SARS-CoV pET-15b-PLpro(1541–1855) (cloned between
the restriction sites BamH I and Bpu1102 I) and the HCoV-NL63 pET-15b-PLP2(1565–1894) (cloned between the restriction sites BamH I and Bpu1102 I) were obtained from Dr. Susan Baker’s lab
at Loyola University Chicago, Stritch School of Medicine. For crystallization,
SARS-CoV PLpro(1541–1855) fused at the N-terminus
to TEV protease cleavage sites and a 6 histidine tag was synthetized
and codon optimized and cloned into pET11a using the restriction sites Nde I and Bpu1102 I by BioBasic Inc. Costar
96-well black microplates were purchased from Corning. Synthetic peptide
substrate, Z-RLRGG-AMC, used for IC50 determination was
purchased from Bachem. The E. coli expression strain
BL21(DE3) was purchased from Novagen. LB medium components were purchased
from BD Biosciences. The 5 mL HiTrap chelating HP column and the Superdex
200 were obtained from GE Healthcare, Life Sciences. Bradford reagent
and BSA standard solution used for quantification of protein concentration
were purchased from Bio-Rad. Human serum albumin (HSA, catalog no.
A9511, purity 97–99%) for serum shift assays was obtained from
Sigma-Aldrich.
the restriction sites BamH I and Bpu1102 I) and the HCoV-NL63 pET-15b-PLP2(1565–1894) (cloned between the restriction sites BamH I and Bpu1102 I) were obtained from Dr. Susan Baker’s lab
at Loyola University Chicago, Stritch School of Medicine. For crystallization,
SARS-CoV PLpro(1541–1855) fused at the N-terminus
to TEV protease cleavage sites and a 6 histidine tag was synthetized
and codon optimized and cloned into pET11a using the restriction sites Nde I and Bpu1102 I by BioBasic Inc. Costar
96-well black microplates were purchased from Corning. Synthetic peptide
substrate, Z-RLRGG-AMC, used for IC50 determination was
purchased from Bachem. The E. coli expression strain
BL21(DE3) was purchased from Novagen. LB medium components were purchased
from BD Biosciences. The 5 mL HiTrap chelating HP column and the Superdex
200 were obtained from GE Healthcare, Life Sciences. Bradford reagent
and BSA standard solution used for quantification of protein concentration
were purchased from Bio-Rad. Human serum albumin (HSA, catalog no.
A9511, purity 97–99%) for serum shift assays was obtained from
Sigma-Aldrich.
3
Fluorescence-based Deubiquitinating Enzyme Assay
4
Enzymatic Assay of Deubiquitinases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Activity assays of DUBs against AMC-labeled substrates were performed using reaction buffer (150 mM NaCl, 20 mM TRIS pH 7.5, 10 mM DTT), 1 µM DUBs (deviating concentrations are indicated in the respective figures and the corresponding legends), 5 µM Ub-AMC (UbiQ-Bio, The Netherlands), 5 µM NEDD8-AMC (Enzo Life Science) or 100 µM zRLRGG-AMC (BACHEM AG, Switzerland). The reaction was performed in black 96-well plates (Corning) at 30 °C and fluorescence was measured using the Infinite F200 Pro plate reader (Tecan) equipped for excitation wavelength of 360 nm and an emission wavelength of 465 nm. The data was collected using Magellan 7.1 software (Tecan). The presented results are means of three independent cleavage assays.
5
Fluorometric Assay for DUB Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Activity assays of DUBs against AMC-labeled substrates were performed using reaction buffer (150 mM NaCl, 20 mM TRIS pH 7.5, 10 mM DTT), 1 µM DUBs, 100 μM zRLRGG-AMC (BACHEM AG, Switzerland) or 5 µM Ub-AMC (UbiQ-Bio, The Netherlands). The reaction was performed in black 96-well plates (Corning) at 30 °C and fluorescence was measured using the Infinite F200 Pro plate reader (Tecan) equipped for an excitation wavelength of 360 nm and an emission wavelength of 465 nm and the data was collected using Magellan 7.1 software (Tecan). The presented results are means of three independent cleavage assays.
6
Deubiquitinating Enzyme Activity Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Activity assays of DUBs against AMC-labeled Ub/UbL substrates were performed using reaction buffer (150 mM NaCl, 20 mM TRIS pH 7, 10 mM DTT) 1 µM DUBs and 10 µM zRLRGG-AMC (BACHEM AG, Switzerland), 1 µM Sumo1-AMC, 1 µM Sumo2-AMC (Boston Biochem, Inc., USA), 1 µM ISG15-AMC (Boston Biochem, Inc., USA), 1 µM Nedd8-AMC (ENZO Life Sciences GmbH, Germany), LC3A-AMC (Boston Biochem, Inc., USA), or 5 µM Ub-AMC (UbiQ-Bio, The Netherlands). The reaction was performed in black 96-well plates (Corning) at 30 °C and released fluorescence was measured using the Infinite F200 Pro plate reader (Tecan) equipped for excitation wavelength of 360 nm and an emission wavelength of 465 nm. The measurements were performed in triplicate and the mean is presented.
7
Biflavone Inhibition of SARS-CoV-2 PLpro
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All of the tested compounds were retrieved from 4 °C storage of NCLTCMs, which were all pure isolates from natural sources. The NCLTCMs accession numbers for biflavones 1 –9 were 3001852, 3000847, 3000889, 3000854, 3000863, 3001926, 3004125, 3004129 and 3003784, respectively. Purities of the biflavones were all greater than 98% as determined by HPLC, and a 1 mg aliquot of each compound was weighed to prepare a 10 mM DMSO stock. Recombinant SARS-CoV-2 papain-like protease (Glu1564-Val1880, Cat. 40593-V07E) and recombinant human pro-ISG15 protein (Met1-Ser165, Cat. 12729-HNAE) were purchased from Sino-Biological Inc (Beijing, China). The fluorogenic substrate Z-RLRGG-AMC was purchased from Bachem (Bubendorf, Switzerland).
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!