Ten microliters of trypsin/chymotrypsin (Sigma Aldrich, Dorset, UK) working solution (0.1 µM in 1 mM HCl) was added into the wells of a black micro-titre plate with corresponding substrate and peptide replicates (0.1–100 µM) in 10 mM phosphate buffer (final volume 210 μL). Additionally, Phe-Pro-Arg- NHMec (Bachem, Cambridge, UK) and Succinyl-Ala-Ala-Pro-Phe-NHMec (Bachem, Cambridge, UK) were performed as substrates for trypsin and chymotrypsin, respectively. The fluorescence intensity of NHMec was monitored at 37 °C continuously for 30 min by a FLUOstar OPTIMA multi-well plate reader (BMG Labtech, Ortenberg, Germany) at wavelengths of 460 nm for emission and 395 nm for excitation. The inhibition curves of different protease were plotted using the Morrison equation and non-linear regression analysis, which were showed as outlined before [26 (link)].
Succinyl ala ala pro phe nhmec
Manufactured by Bachem
Sourced in United Kingdom
Succinyl-Ala-Ala-Pro-Phe-NHMec is a tetrapeptide substrate used for the fluorometric detection and measurement of chymotrypsin-like protease activity. It is a concise and well-defined compound that can be utilized in various research and diagnostic applications.
Automatically generated - may contain errors
5 protocols using succinyl ala ala pro phe nhmec
1
Fluorometric Enzymatic Assay for Proteases
2
Enzymatic Inhibition Assays for Proteases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Trypsin and chymotrypsin inhibition assays were performed as previously described [24 (link)]. The substrate of trypsin and chymotrypsin were Phe-Pro-Arg-AMC and Succinyl-Ala-Ala-Pro-Phe-NHMec, respectively, and obtained from Bachem, Merseyside, UK. In the tryptase inhibition assay, peptide samples were first dissolved in tryptase assay buffer pH 7.6, containing 0.05 M Tris, 0.15 M NaCl and 0.2% (w/v) PEG 6000 (final volume 210 μL) to obtain different concentrations of 0.5, 1, 2 and 4 mM and then added to the wells of a black 96-well plate containing (Boc-Phe-Ser-Arg-NHMec, obtained from Bachem, Merseyside, UK) (50 μM). Before measurement, tryptase (2.5 μL from 1 mg/mL stock solution, Calbiochem, Nottingham, UK) was added to each well. The rate of hydrolysis of substrate was monitored continuously at 37 °C and 460 nm with a Fluostar Optima plate reader (BMG Labtech GmbH, Offenburg, Germany)
3
Peptide Inhibition of Trypsin and Chymotrypsin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The inhibitory activity of peptides towards trypsin and chymotrypsin was tested the same as detailed in Wang’s study [11 (link)]. Peptides were initially prepared in a range of 10−3–10−6 M and then diluted to 16 concentrations. Each dilution was tested in duplicates. Additionally, Phe-Pro-Arg-AMC and Succinyl-Ala-Ala-Pro-Phe-NHMec (obtained from Bachem, UK) were served as substrates for trypsin and chymotrypsin, respectively.
4
Fluorometric Enzyme Kinetics Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Trypsin (10 μl from 0.1 μM stock solution in 1 mM HCl), was added to the wells of a microtitre plate containing substrate (Phe-Pro-Arg-NHMec) (50 μM) and synthetic peptide replicates (0.1–100 μM) in 10 mM phosphate buffer, pH 7.4, containing 2.7 mM KCl and 137 mM NaCl (final volume 210 μl).
Chymotrypsin (10 μl from 0.1 μM stock solution in 1 mM HCl) was added to the wells of a microtitre plate containing substrate (Succinyl–Ala–Ala–Pro–Phe–NHMec, obtained from Bachem, U.K.) (50 μM) and synthetic peptide replicates (0.1–100 μM) in 10 mM phosphate buffer, pH 7.4, containing 2.7 mM KCl and 137 mM NaCl (final volume 210 μl).
Tryptase (2.5 μl from 1 mg/ml stock solution, Calbiochem, U.K.), was added to the wells of a microtitre plate containing substrate (Boc-Phe-Ser-Arg-NHMec, obtained from Bachem, U.K.) (50 μM) and synthetic peptide replicates (0.5, 1, 2 and 4 mM) in tryptase assay buffer, pH 7.6, containing 0.05 M Tris, 0.15 M NaCl and 0.2% (w/v) PEG 6000 (final volume 210 μl).
Each determination was carried out in triplicate. The rate of hydrolysis of substrate was monitored continuously, at 37°C, by measuring the rate of increase in fluorescence due to production of 7-amino-4-methylcoumarin (NH2Mec) at 460 nm (excitation 360 nm) in a CytoFluor® multi-well plate reader Series 4000 spectrofluorimeter.
Chymotrypsin (10 μl from 0.1 μM stock solution in 1 mM HCl) was added to the wells of a microtitre plate containing substrate (Succinyl–Ala–Ala–Pro–Phe–NHMec, obtained from Bachem, U.K.) (50 μM) and synthetic peptide replicates (0.1–100 μM) in 10 mM phosphate buffer, pH 7.4, containing 2.7 mM KCl and 137 mM NaCl (final volume 210 μl).
Tryptase (2.5 μl from 1 mg/ml stock solution, Calbiochem, U.K.), was added to the wells of a microtitre plate containing substrate (Boc-Phe-Ser-Arg-NHMec, obtained from Bachem, U.K.) (50 μM) and synthetic peptide replicates (0.5, 1, 2 and 4 mM) in tryptase assay buffer, pH 7.6, containing 0.05 M Tris, 0.15 M NaCl and 0.2% (w/v) PEG 6000 (final volume 210 μl).
Each determination was carried out in triplicate. The rate of hydrolysis of substrate was monitored continuously, at 37°C, by measuring the rate of increase in fluorescence due to production of 7-amino-4-methylcoumarin (NH2Mec) at 460 nm (excitation 360 nm) in a CytoFluor® multi-well plate reader Series 4000 spectrofluorimeter.
5
Chymotrypsin Inhibition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Inhibitory activity assays on synthetic peptide replicates and their various P1-sitesubstituted variants against chymotrypsin, were performed exactly as detailed for the trypsin inhibition assay, except that the target protease was chymotrypsin and the fluorogenic substrate utilised was Succinyl-Ala-Ala-Pro-Phe-NHMec (obtained from Bachem, UK).
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!