Bovine serum albumin (> 99.9%, probumin, crystallized, 810014, lot number 121, Merck KGaA, Darmstadt, Germany), chicken avidin (98%, 786-582, VWR International GmbH, Darmstadt, Germany), myoglobin from horse skeletal muscle (M0630-250MG, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), jacalin (> 95%, SRP6176-5MG, Sigma-Aldrich Chemie GmbH), bovine apotransferrin (> 95%, PSB-PRO-511, Biozol Diagnostics), and recombinant protein G (> 96%, PSB-PRO-402, Biozol Diagnostics, Munich, Germany) were used. The proteins were dissolved in pure water (Barnstead Gen-Pure xCAD plus water purification system, Thermo Scientific, Braunschweig, Germany) to an expected concentration of 1 mg/mL (gravimetrically), and 500-μL aliquots were stored at − 20 ∘C for further analysis. Commercial birch pollen extract (Allergopharma GmbH & Co. KG, Hamburg, Germany) was provided by courtesy of I. Bellinghausen (University Clinics, Mainz, Germany). NIST reference materials (total protein standard, SRM 927e - Bovine Serum Albumin) and amino acids (SRM 2389a - Amino Acids) were used. The characterization of the test proteins is shown in the Electronic Supplementary Material: Protein Characterization.
M0630 250mg
Manufactured by Merck Group
Sourced in Germany
M0630-250MG is a laboratory reagent product offered by Merck Group. It is a 250 milligram quantity of a specific chemical compound. The core function of this product is to serve as a research material for scientific investigations, but its intended use is not provided in this factual description.
Automatically generated - may contain errors
4 protocols using m0630 250mg
1
Protein Characterization and Quantification
2
Protein Standards for Analytical Validation
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The protein standards were all purchased from Sigma and included: α-casein (α-CN 23.6 kDa) from bovine milk (C6780-250MG, 70% pure), β-lactoglobulin (β-LG, 18.7 kDa) from bovine milk (L3908-250MG, 90% pure), albumin from bovine serum (BSA, 66.5 kDa, A7906-10G, 98% pure), and myoglobin from horse skeletal muscle (Myo, 16.9 kDa, M0630-250MG, 95–100% pure and salt-free) [28 (link),29 (link)].
3
Antioxidant Capacity Measurement in Plasma
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TAS measures the capacity of plasma samples to counteract a redox reaction induced by free radicals (Miller et al. 1993; Cohen et al. 2007 ) and is primarily the result of the pooled effect of all extracellular antioxidant compounds of the blood (Costantini 2011). TAS was analyzed as described in López-Arrabé et al. (2014b) . As standard for the assays we used Trolox (a watersoluble a-tocopherol derivative), and TAS levels are expressed in Trolox-equivalent units. The assays were run on a Synergy HT multimode microplate reader (BioTek Instruments, Winooski, VT). To accurately control the reaction time, only one column of the plate was used at a time. To standards and samples were sequentially added metmyoglobin (a mix of equal volumes of myoglobin [M0630-250MG; Sigma-Aldrich, St. Louis, MO] and potassium ferricyanate), ABTS (the chromogen 2,2 0 -azinobis-(3ethylbenzothiazoline-6-sulphonic acid), and H 2 O 2 , which started the reaction. Kinetic measurements were immediately started, recording absorbance at 660 nm every 5 s. The temperature was maintained at 377C during assays. All samples were assayed in duplicate, and results showed high repeatability (r p 0:981, N p 234, P < 0:001) and an interassay CV of 3.63%.
4
Trolox-Equivalent Antioxidant Capacity
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TAS was analyzed as described by López-Arrabé et al. (2014b) . As standard for the assays, we used Trolox (a water-soluble a-tocopherol derivative), and TAS levels are expressed in Trolox-equivalent units. The assays were run on a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT). In order to accurately control the reaction time, only one column of the plate was used at a time. To standard or samples were sequentially added metmyoglobin (a mixed of equal volumes of myoglobin [M0630-250MG, Sigma-Aldrich, St. Louis] and potassium ferricyanate), ABTS (the chromogen, 2,2 0 -azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)), and H 2 O 2 , starting the reaction. Kinetic measurements were immediately started, recording absorbance at 660 nm every 5 s. The temperature was maintained at 377C during assays. All samples were assayed in duplicate, and results showed a high repeatability (r p 0.981, N p 234, P ! 0.001) and an interassay CV of 3.63%.
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