BSM (Type I-S) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and treated with N-glycosidase (PNGase F; New England Biolabs, MA, USA) according to the manufacturer’s instructions. The released N-glycans were separated from the remaining peptides and salts with a graphitized carbon cartridge [30 (link)] and derivatized with ProA (Sigma-Aldrich) [31 (link)]. Briefly, a ProA-derivatizing reagent made with sodium cyanoborohydride (9.7 mg) and ProA (16.2 mg) in 150 µl of 30% acetic acid in dimethyl sulfoxide (v/v) was added to the released N-glycans. Excess fluorophores were removed using microcrystalline cellulose (Sigma-Aldrich, St. Louis, MO, USA) column chromatography.
Bsm type 1 s
Manufactured by Merck Group
Sourced in Denmark, United States
The Merck Group's BSM (Type I-S) is a versatile laboratory equipment designed for general-purpose applications. It functions as a benchtop stirring system, providing efficient mixing and stirring capabilities for a variety of laboratory samples and solutions.
Automatically generated - may contain errors
5 protocols using bsm type 1 s
1
Derivatization of N-Glycans using Prolinol (ProA)
2
Characterization of BSM (Type I-S) Purity
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BSM (Type I-S) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The purity was evaluated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% polyacrylamide gel, as previously described [16 (link)]. Molecular mass markers were purchased from Bio-Rad Laboratories (Hercules, CA, USA), and the gel was stained with Coomassie blue R-250.
3
Bovine Milk Protein pH-Dependent Behavior
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BLG from bovine milk, BSM (Type I-S), and PGM (Type III) were purchased from Sigma Aldrich (Brøndby, Denmark), and were used as received. Protein solutions were prepared by dissolving proteins in 100 mM PBS solutions. By addition of HCl as appropriate, the pH values of the buffer solutions were adjusted to pH 7, 5, and 3 where the BLG is negatively, neutral, and positively charged, respectively [4 (link)]. All buffer solutions were filtered (polyethersulfone 0.20 mm).
4
Bovine Milk Protein Interactions
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BLG from bovine milk and BSM (Type I-S) were purchased from Sigma Aldrich (Sigma Aldrich A/S, Brøndby, Denmark), and were used as received. Protein solutions with the concentration of 1 mg/mL were prepared by dissolving proteins in 10 mM phosphate buffered saline (PBS) solutions.
The pH values of the buffer solutions were adjusted to 7.4, 5.0, and 3.0 by the addition of HCl or NaOH as appropriate. For the mixtures of BSM and BLG, the two protein solutions were mixed at the ratio of 1:1 (v:v), while the final concentrations of the total proteins were set at 1 mg/mL or 2 mg/mL. The concentration of 1 mg/mL was used for the first mixture (MIX 1) where each protein (BLG and BSM) has 0.5 mg/mL in concentration. The second mixture (MIX 2) was prepared with 1 mg/mL concentration of each proteins (BLG and BSM), hence with 2 mg/mL in total protein concentration.
The pH values of the buffer solutions were adjusted to 7.4, 5.0, and 3.0 by the addition of HCl or NaOH as appropriate. For the mixtures of BSM and BLG, the two protein solutions were mixed at the ratio of 1:1 (v:v), while the final concentrations of the total proteins were set at 1 mg/mL or 2 mg/mL. The concentration of 1 mg/mL was used for the first mixture (MIX 1) where each protein (BLG and BSM) has 0.5 mg/mL in concentration. The second mixture (MIX 2) was prepared with 1 mg/mL concentration of each proteins (BLG and BSM), hence with 2 mg/mL in total protein concentration.
5
Interactions of Bovine Milk Proteins
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BLG from bovine milk, BSM (Type I-S), and PGM (Type III) were purchased from Sigma-Aldrich (Brøndby, Denmark), and were used as received. Protein solutions with the concentration of 1 mg/mL were prepared by dissolving in 10 mM phosphate buffered saline (PBS) solutions and were used throughout the study. The pH values of the buffer solutions were adjusted to 7.4, 5, and 3 by addition of HCl or NaOH. For the mixture of BLG and mucins, the two protein solutions were mixed directly at the ratio of 1:1 (v:v) and the total protein concentrations were remained at either 1 mg/mL. Due to relatively weaker lubricating capabilities of PGM at 1 mg/mL (Lee, Müller, Rezwan, & Spencer, 2005) , PGM, BLG, and PGM-BLG mixed solutions were studied at 10 mg/mL too.
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