Heat-inactivated fetal bovine serum (FBS, HyCylone™) and PBS (10X), pH 7.4 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco’s modified Eagle’s medium high glucose 4.5 g/L (DMEM), penicillin-streptomycin solution, trypsin-EDTA (0.25%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS substrate), 3,3′,5,5′-tetramethylbenzidine (TMB substrate), human glycated albumin, elastase from human leukocytes, N-succinyl-Ala-Ala-Ala-p-nitroanilide, oleanolic acid, hyaluronidase from bovine testes, 4 (dimethylamino)benzaldehyde (DMAB), hyaluronic acid sodium salt from cockscomb, sodium aurothiomalate hydrate (SATMH) and Collagenase Activity Colorimetric Assay Kit (MAK293) were purchased from Merck (Waltham, MA, USA). ELISA Human IL-6 Kit (900-K16) and ELISA Human IL-8 Kit (900-K18) were purchased from PeproTech (London, UK). Human Total MMP-1 DuoSet ELISA (DY901B-05), Human MMP-2 Duo-Set ELISA (DY902), and Human Pro-Collagen I alpha 1 DuoSet ELISA (DY6220-05) were purchased from R&D Systems (Minneapolis, MI, USA).
N succinyl ala ala ala p nitroanilide
Manufactured by Merck Group
Sourced in United States, Germany, Italy
N-succinyl-Ala-Ala-Ala-p-nitroanilide is a synthetic substrate that is used for the enzymatic detection and quantification of proteases, specifically those that cleave peptide bonds after alanine residues. This substrate is commonly used in biochemical and enzymological research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (9 mentions)
Oleanolic acid, by Merck Group (7 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (7 mentions)
Gallic acid, by Merck Group (7 mentions)
Quercetin, by Merck Group (7 mentions)
Ascorbic acid, by Merck Group (7 mentions)
Porcine pancreatic elastase, by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Kojic acid, by Merck Group (5 mentions)
Trizma base, by Merck Group (5 mentions)
L tyrosine, by Merck Group (4 mentions)
Elastase from porcine pancreas, by Merck Group (4 mentions)
Epigallocatechin gallate (egcg), by Merck Group (4 mentions)
Hyaluronidase, by Merck Group (4 mentions)
N 3 2 furyl acryloyl leu gly pro ala, by Merck Group (4 mentions)
Hydrochloric acid (hcl), by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Rutin, by Merck Group (3 mentions)
Mushroom tyrosinase, by Merck Group (3 mentions)
Tyrosinase from mushroom, by Merck Group (3 mentions)
51 protocols using n succinyl ala ala ala p nitroanilide
1
Evaluation of Skin Inflammation Markers
2
Antioxidant and Enzymatic Assays
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2, 2-Diphenyl-1-picrylhydrazyl (DPPH), 2-propanol, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), arbutin, crocin, epigallocatechin gallate (E), N-Succinyl-Ala-Ala-Ala-p-Nitroanilide, porcine pancreatic elastase (PPE) and tyrosinase, were purchased from Merck, Milan, Italy. Ethanol, methanol, dimethyl sulfoxide (DMSO), hydrogen peroxide (H2O2) and sodium hydroxide (NaOH) were purchased from Carlo Erba Reagents, Milan, Italy. Hydrochloric acid (HCl) and ferric sulphate (Fe2(SO4)3) were bought from VWR chemicals, Milan, Italy. All the cell culture reagents were purchased from Euroclone, Milan, Italy.
3
Equine Lyosecretome's Anti-Elastase Activity
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The in vitro inhibitory effect of equine Lyosecretome on the elastase enzyme was evaluated using the method previously described in the literature, with some modifications [49 (link)]. Pancreatic porcine elastase (Merck Life Science S.r.l.) was solubilized in phosphate buffer pH 6.8 (0.5 IU mL−1). The substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide (Merck Life Science S.r.l.) was dissolved in TRIS buffer until a final concentration of 0.41 mmol L−1 was reached. All the tested concentrations (2, 5, 10, 20 mg mL−1) were incubated with the enzyme for 20 min, and, consequently, the substrate was added right before reading the microplates to begin the reaction. The kinetic reaction was monitored by spectrophotometric analysis (Synergy HT) at the absorbance of 410 nm for 35 min (measurements were made every minute). The reaction mixture in the absence of sample was used as a negative control, while the epigallocatechin gallate (EGCG) (Merck Life Science S.r.l.) was used as a positive control. The absorbance value of each sample was subtracted from the absorbance of the blank mixture (sample without the enzyme and substrate). Analyses were performed in triplicate. The inhibition rate was reported as a percentage of anti-elastase activity and calculated as follows:
where ACTR is the negative control absorbance and Asamp is the Lyosecretome sample absorbance.
where ACTR is the negative control absorbance and Asamp is the Lyosecretome sample absorbance.
4
Melanoma Cell Lines Characterization
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A375 (ATCC CRL-1619) human malignant melanoma, SH-4 (ATCC CRL-7724) human melanoma, and B16-F10 (ATCC CRL-6475) murine melanoma cell lines were purchased from LGC Standards (Łomianki, Poland). Immortalized human keratinocytes HaCaT were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany). Fetal bovine serum (FBS) was obtained from Pan-Biotech (Aidenbach, Germany). Dulbecco’s modified Eagle’s medium (DMEM)/high glucose, with and without phenol red, Dulbecco’s phosphate buffered saline (DPBS), mushroom tyrosinase from Agaricus bisporus, L-tyrosine, 3,4-dihydroxy-l -phenylalanine (L-DOPA), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2′,7′-dichlorofluorescin diacetate (H2DCFDA), N-acetylcysteine (NAC), N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), kojic acid (≥99.0%), chlorogenic acid (≥95%), ellagic acid (≥95%), gallic acid (97.5–102.5%), kaempferol (≥97.0%), quercetin (≥95.0%), rutin (≥94.0%), 1,10-phenantroline (≥99.0%), and neutral red solution (3.3 g/L) were purchased from Merck (Darmstadt, Germany). Water (H2O), formic acid (HCOOH), and acetonitrile (CH₃CN) for HPLC analysis were purchased from J.T. Baker (Witko, Łódź, Poland).
5
Enzyme Inhibition Assays for Plant Extracts
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The elastase inhibitory activity of the prioritized extract was evaluated using elastase from porcine pancreas (PPE) type IV (Merck KGaA, Darmstadt, Germany) and the substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide (Merck KGaA, Darmstadt, Germany), as previously described [36 (link)]. The capacity of the extract to inhibit the catalytic action of tyrosinase in the oxidation of L-DOPA to dopachrome was determined, as previously reported [37 (link)].
6
Development and Characterization of Copper Tripeptide Formulations
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The copper tripeptide (SpecPed-GCu11P, Spec-Chem Industry Inc., Nanjing, China) was of cosmetic grade and was kindly supplied by Alfa Sagittarius (Kraków, Poland). Hydrogenated lecithin (Emulmetik 950, Lucas Meyer Cosmetics, Massy, France) was kindly supplied by Naturallia Sp. z o.o. (Brzeg, Poland). Dicetyl phosphate, N-Succinyl-Ala-Ala-Ala-p-nitroanilide, elastase from porcine pancreas, mushroom tyrosinase and L-DOPA were all purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Stearylamine (97%) and cholesterol (95%) were purchased from Alfa Aesar (Thermo Fisher (Kandel) GmbH, Kandel, Germany). From Pol-Aura (Zawroty, Poland), 0.1 M Tris-HCl buffer was purchased. All other chemicals, such as potassium dihydrogen phosphate (POCh, Gliwice, Poland), sodium hydrogen phosphate (POCh), methanol (Chempur, Piekary Śląskie, Poland) and acetonitrile (Chempur), were of analytical grade. Spectra/Por 1 regenerated cellulose membrane with a 6–8 kDa molecular weight cut-off (MWCO), which was used in the microdialysis tests, was purchased from Spectrum Laboratories Inc., (DG Breda, The Netherlands). The deionized water used in all formulations was additionally filtered through a Milli-Q system.
7
Isolation and Purification of Rice Biopesticides
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The extraction and isolation solvents were purchased from Junsei Chemical Co., Ltd., Tokyo, Japan and Fisher Scientific company, Hampton, NH, USA. Chemicals for antioxidant assays were acquired from Fujifilm Wako Pure Chemical Corporation, Osaka, Japan. Elastase from porcine pancreas Type IV, tyrosinase from mushroom lyophilized powder, N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), L-tyrosine, oleanolic acid, vanillin, myricetin, and all buffer components were acquired from Sigma-Aldrich, St. Louis, MO, USA.
Pure momilactones A, B, and tricin were isolated previously from rice husk by our laboratory [12 (link)]. Briefly, the ethyl acetate (EtOAc) extract of 7 kg Koshihikari husks was separated by open column chromatography over silica gel with the mobile phase as combinations of hexane and EtOAc. Momilactones A and B were isolated from the eluant hexane:EtOAc (8:2) by a repeated column chromatography while tricin (a yellow powder) was purified from the last fractions of the eluant hexane: EtOAc (7:3). The identification of such pure compounds is described in detail in the following parts.
Pure momilactones A, B, and tricin were isolated previously from rice husk by our laboratory [12 (link)]. Briefly, the ethyl acetate (EtOAc) extract of 7 kg Koshihikari husks was separated by open column chromatography over silica gel with the mobile phase as combinations of hexane and EtOAc. Momilactones A and B were isolated from the eluant hexane:EtOAc (8:2) by a repeated column chromatography while tricin (a yellow powder) was purified from the last fractions of the eluant hexane: EtOAc (7:3). The identification of such pure compounds is described in detail in the following parts.
8
Evaluation of Elastase Activity in Fibroblasts
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Proliferating normal human diploid fibroblasts (BJ cells) were cultured as described before [35 (link)]. In order to evaluate elastase activity, the amount of released p-nitroaniline, which was hydrolyzed from the substrate (N-succinyl-Ala-Ala-Ala-p-nitroanilide) by elastase, was determined by measuring the absorbance at 405 nm. Thus, BJ fibroblasts were seeded into 96-well microplates and the next day they were treated with compounds 1 to 7 at a final concentration of 5 μM for each compound and of 1 μg/mL or 10 μg/mL of concentration for each extract, for 24 h. Afterwards, cells were lysed in 100 mM Tris-HCl (pH 7.6) with 0.1% Triton X-100 buffer and subsequently, 2 mM N-succinyl-Ala-Ala-Ala-p-nitroanilide (Sigma-Aldrich) was added to each well followed by incubation at 37 °C for 1 h. The absorbance at 405 nm was measured using a microplate reader Infinite 200 PRO series (Tecan Trading AG, Switzerland).
9
Antioxidant and Enzymatic Activity Assays
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Ascorbic acid, collagenase from Clostridium histolyticum, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), elastase from porcine pancreas, (-)-epigallocatechin gallate (EGCG), Folin–Ciocalteu reagent, tyrosinase from mushroom (≥1000 unit/mg solid), ethylenediaminetetraacetic acid, disodium dihydrate (Na2EDTA*2H2O), Levodopa (L-DOPA), N-[3-(2-Furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA), N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), Tricine (≥99%; titration) were obtained from Sigma-Aldrich (Steinheim, Germany). Phosphate-buffered saline (PBS) was purchased from Gibco (Carlsbad, CA, USA). Reference substances were from ChromaDex (Irvine, CA, USA). Acetonitrile, formic acid and water for LC analysis were from Merck (Darmstadt, Germany). All others chemicals were of analytical grade and were obtained from Polish Chemical Reagent Company (POCH, Gliwice, Poland).
10
Antioxidant and Enzymatic Inhibition Assays
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Folin–Ciocalteu’s phenol reagent, aluminum chloride (AlCl3), dimethyl sulfoxide (DMSO), sodium acetate (NaOAc), quercetin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), L-ascorbic acid, potassium persulfate (K2S2O8), elastase from porcine pancreas, epigallocatechin gallate (EGCG), N-succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), tyrosinase from mushroom, kojic acid (KA), and 3,4-dihydroxy-L-phenylalanine (L-DOPA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium carbonate (Na2CO3) was purchased from Merck (Darmstadt, Germany). Gallic acid was purchased from TCI America (Portland, OR, USA). Dipotassium phosphate (K2HPO4) and monobasic potassium phosphate (KH2PO4) were purchased from HiMedia (Mumbai, India). Tris base was purchased from Vivantis Technologies (Shah Alam, Malaysia). Ethanol and methanol were purchased from RCI Labscan (Bangkok, Thailand). All chemicals and reagents were analytical grades.
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