Tricine buffer

Manufactured by Merck Group

Sourced in United States

Tricine buffer is a laboratory reagent used to maintain a stable pH environment in various biochemical applications. It provides buffering capacity in the pH range of 7.0-9.0, making it suitable for a variety of protein-based experiments and analyses.

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Tricine (85 citations)

7 protocols using tricine buffer

Collagenase Inhibition Assay

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Reaction mixture
(total volume 150 μL): 125 μL of collagenase from Hathewayahistolytica(formerly C. histolyticum (0.8 mg/mL) dissolved in 50 mM tricine
buffer (Sigma-Aldrich) was mixed with 12.5 μL of tested sample
(0.8 mg/mL) dissolved in DMSO. After 15 min of incubation at 25 °C,
12.5 μL of FALGPA (N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala)
(9.6
mM) dissolved in 50 mM Tricine buffer (Sigma-Aldrich) was added. The
absorbance (λ 340 nm, 25 °C) of the tested sample (A sample)
started to be measured exactly 1 min after the addition of the FALGPA
and continued for 15 min, using a microplate reader (Synergy HT) in
96-well microtiter plates. The experiment was done in quadruplicate.
Chlorogenic acid was used as a positive control and DMSO as an NC
(A control). The blank solution (A blank) consisted of tricine buffer
and tested sample or DMSO (in the case of a NC). The collagenase inhibitory
activity was calculated according to the formula
Statistical analysis was carried out
using GraphPad Prism 6.01 software. Results are expressed as the mean
± standard deviation. Statistical analyses were performed using
the nonparametric (n < 30) Kruskal–Wallis
analysis, together with Dunn’s posthoc test. Values of p less
than 0.05 were considered statistically significant; n = number of repetitions. ** indicates a significant difference p < 0.01; ***p < 0.001 vs NC - DMSO.
Values are mean ± SD; n = 4.

Vasilev H., Šmejkal K., Jusková S., Vaclavik J, & Treml J. (2024). Five New Tamarixetin Glycosides from Astragalus thracicus Griseb. Including Some Substituted with the Rare 3-Hydroxy-3-methylglutaric Acid and Their Collagenase Inhibitory Effects In Vitro. ACS Omega, 9(16), 18023-18031.

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Microsomal Preparation and Nicotine Metabolism

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Microsomes were prepared from flash-frozen tissues. Five mice were used for each sex, strain and condition. Tissues were homogenized in ice-cold potassium phosphate buffer pH 7.4, 6x with a glass-teflon homogenizer, and centrifuged 20 min at 9,000 g. The S9 fraction was centrifuged 60 min at 100,000 g to obtain a microsomal pellet which was resuspended, further homogenized 6x, centrifuged another 60 min at 100,000 g and finally resuspended and homogenized 6x in potassium phosphate buffer containing 20% glycerol for storage at −80°C. Prior to freezing, protein concentrations were determined by the Bradford Protein assay. All incubations were carried out in 96-well PCR plates in 50μl total volume at 37 ºC, as previously described (Teitelbaum et al., 2017 ) 10 minutes in tricine buffer containing 1 mM NADPH (Sigma-Aldrich) and 100 μM nicotine (Toronto Research Chemicals, Toronto Canada). Reactions were quenched by adding 10μl 15% zinc sulfate containing 200ng/ml of the internal standard, (d₃)-nicotine-N-oxide (Toronto Research Chemicals, Toronto Canada). Samples were centrifuged and supernatants removed for LC/MS analysis.

Bloom A.J., Upadhyaya P, & Kharasch E.D. (2019). Strain-specific Altered Nicotine Metabolism in 3,3′-diindolylmethane (DIM) Exposed Mice. Biopharmaceutics & drug disposition, 40(5-6), 188-194.

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Enzymatic Assays for Collagenase, Elastase, and Tyrosinase

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Tricine buffer, collagenase (ChC—EC.3.4.23.3), N-[3-(2-furyl) acryloyl]-Leu–Gly–Pro–Ala (FALGPA), EGCG, N-Succinyl-Ala–Ala–Ala-p-nitroanilide (AAAPVN), Tris-HCL buffer, pancreatic elastase (PE), L-DOPA, mushroom tyrosinase, phosphate buffer, Kojic acid, calcium chloride, hyaluronic acid, potassium metaborate (KBO_2), hyaluronidase, acetate buffer, 10 N HCl, acetic acid, and p-dimethylaminobenzaldehyde (DMAB) were bought from Sigma-Aldrich (Heliopolis, Cairo, Egypt). Methanol and hexane were bought from Al-brouj (Giza, Egypt). All solvents used were of analytical grade.

Younis M.M., Ayoub I.M., Mostafa N.M., El Hassab M.A., Eldehna W.M., Al-Rashood S.T, & Eldahshan O.A. (2022). GC/MS Profiling, Anti-Collagenase, Anti-Elastase, Anti-Tyrosinase and Anti-Hyaluronidase Activities of a Stenocarpus sinuatus Leaves Extract. Plants, 11(7), 918.

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In Vitro Assessment of Collagenase Inhibition by Fenugreek Seed Powder

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Fenugreek seed powder was received from Herbal Acharn’s Home Co., Ltd. (Bangkok, Thailand). Rutin trihydrate (>98% purity), tricine buffer, collagenase from clostridium histolyticum and FALGPA (N-[3(2-furyl) acryloyl]-Leu-Gly-Pro-Ala), direct red 80, picric acid, and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, ethyl acetate, absolute ethanol, formic acid, sodium dehydrate phosphate, disodium hydrogen phosphate, hydrochloric acid, and sodium hydroxide were purchased from Carlo Erba (Emmendingen, Germany). Commercial grade ethanol was purchased from Italmar Co., Ltd. (Bangkok, Thailand). Cholesterol was purchased from Cosmeplus Co., Ltd. (Bangkok, Thailand). Propylene glycol and solubilizer mixture were purchased from S. Tong Chemicals Co., Ltd. (Nonthaburi, Thailand). Sorbitan oleate was purchased from Croda Co., Ltd. (Bangkok, Thailand). Phospholipid (Phospholipon 90G) was purchased from Cargill Siam Ltd. (Bangkok, Thailand). Tocopherol acetate was purchased from Namsiang Co., Ltd. (Bangkok, Thailand). The preservative was purchased from Forecus Co., Ltd. (Bangkok, Thailand). Paraformaldehyde was purchased from Himadia Laboratories (Mumbai, India) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was purchased from Calbiochem (Burlington, MA, USA).

Eaknai W., Bunwatcharaphansakun P., Phungbun C., Jantimaporn A., Chaisri S., Boonrungsiman S., Nimmannit U, & Khongkow M. (2022). Ethanolic Fenugreek Extract: Its Molecular Mechanisms against Skin Aging and the Enhanced Functions by Nanoencapsulation. Pharmaceuticals, 15(2), 254.

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Collagenase and Elastase Assay Protocol

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Collagenase from Clostridium histolyticum, neutrophil elastase, tricine buffer, N-[3-(2-Furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA), N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), mangiferin, oleanolic acid were sourced from Sigma Chemical Co. (St. Louis, MO, USA), and TRIS-HCl, NaCl, CaCl₂, DMSO were sourced from Avantor Performance Materials Poland S.A.

Ochocka R., Hering A., Stefanowicz–Hajduk J., Cal K, & Barańska H. (2017). The effect of mangiferin on skin: Penetration, permeation and inhibition of ECM enzymes. PLoS ONE, 12(7), e0181542.

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Enzymatic and Antioxidant Assays for Bioactive Compounds

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Collagenase from Clostridium histolyticum, neutrophil elastase, tricine buffer, N-[3-(2-Furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA), N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), kojic acid, mangiferin, isomangiferin, hesperidin, vicenin-2, oleanolic acid, hyaluronidase (hyaluronidase from bovine testes, 400–1000 U/mg), tyrosinase (tyrosinase from mushroom), 3-(3,4-Dihydroxyphenyl)-L-alanine (L-DOPA), phosphate buffer (0.175 mM, pH 6.8), hyaluronic acid, bovine serum albumin (BSA), DPPH (2,2-diphenyl-1-picrylhydrazyl), TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine), DMSO (dimethyl sulfoxide), ascorbic acid and phosphoric acid were sourced from the Sigma Chemical Co. (St. Louis, MO, USA), and TRIS-HCl, HCl (77 mM), NaCl, CaCl₂, acetate buffer (acetic acid 79 mM, sodium acetate 24 mM, pH 3.75)—for enzymatic assays—and acetate buffer (0.3 M, pH 3.6)—for FRAP assays—were sourced from Avantor Performance Materials Poland S. A. FeCl₃ × 6 H₂O and HPLC-grade methanol and ethanol were purchased from P.O.Ch. (Gliwice, Poland).

Hering A., Stefanowicz-Hajduk J., Gucwa M., Wielgomas B, & Ochocka J.R. (2023). Photoprotection and Antiaging Activity of Extracts from Honeybush (Cyclopia sp.)—In Vitro Wound Healing and Inhibition of the Skin Extracellular Matrix Enzymes: Tyrosinase, Collagenase, Elastase and Hyaluronidase. Pharmaceutics, 15(5), 1542.

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Collagenase Inhibitory Activity Assay

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Anti-collagenase activity of EERR and EERP was assessed by evaluating the inhibition of collagenase, following the procedure of Wittenuer et al. 14 10 μL collagenase from Clostridium histolyticum (0.01 U/mL, Sigma), 60 μL of 50 mM Tricine buffer pH 7.5 (containing 10 mM CaCl 2 (Merck) and 400 mM NaCl (TCL Co., Ltd)), and 30 μL of extract (7.81 -250 μg/mL) were mixed and incubated at 37°C for 20 min. After incubation, 20 μL of 1 mM N-[3-(2-Furyl)acryloyl]-leu-gly-Pro-Ala (FALGPA, Sigma) in Tricine buffer (Sigma) was added to the mixture. The collagenase inhibitory activities were measured at 335 nm by monitoring for 20 min after starting the reaction. The inhibition value was calculated according to Eq. ( 1). IC 50 values were determined from dose-effect curves.

Elfitriani E., Raif A., Ginting C.N., & Ikhtiari R. (2020). Evaluation of antioxidant and anti-collagenase activity of Rosa damascena L. flower petal and receptacle extract. F1000Research, 9, 716-716.

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