One mg ml-1 of milk powder (Mlekara d.o.o., Pancevo, Serbia), β-lactoglobulin, and mucin (Sigma) were resuspended in gastric juice at pH 2.0 (125 mM NaCl, 7 mM KCl, 45 mM NaHCO3 and 0.3% pepsin). Skim milk was prepared according to the manufacturer’s instructions at 110 mg ml-1. Lactobacilli strains from an MRS overnight culture were harvested by centrifugation (4500 ×g, 10 min), washed twice in 0.85% NaCl (Merck) and resuspended in 10 ml of an appropriate gastric juice containing milk proteins, β-lactoglobulin, skim milk or mucin. The mixtures were incubated anaerobically for 2 h at 37°C. The tolerance of the tested strains was calculated as described in previous section.
β lactoglobulin
Manufactured by Merck Group
Sourced in United States, Germany, Italy, United Kingdom, Sao Tome and Principe, Ireland
β-lactoglobulin is a protein found in the whey fraction of milk. It is commonly used in laboratory settings as a model protein for various studies, including structural analysis, protein folding, and binding interactions. The core function of β-lactoglobulin is to serve as a reference material for these types of research applications.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (26 mentions)
α lactalbumin, by Merck Group (15 mentions)
β casein, by Merck Group (8 mentions)
Sodium chloride (nacl), by Merck Group (6 mentions)
Lysozyme, by Merck Group (5 mentions)
Myoglobin, by Merck Group (4 mentions)
Cytochrome c, by Merck Group (4 mentions)
Thyroglobulin, by Merck Group (3 mentions)
Concanavalin a (cona), by Merck Group (3 mentions)
Transferrin, by Merck Group (3 mentions)
Human serum albumin (hsa), by Merck Group (3 mentions)
Ubiquitin, by Merck Group (3 mentions)
Mucin, by Merck Group (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
N α acetyl l lysine, by Merck Group (2 mentions)
Alcohol dehydrogenase, by Merck Group (2 mentions)
Calcium chloride, by Merck Group (2 mentions)
Potassium chloride (kcl), by Merck Group (2 mentions)
Cholesterol, by Merck Group (2 mentions)
Uric acid, by Merck Group (2 mentions)
72 protocols using β lactoglobulin
1
Gastric Tolerance of Lactobacilli Strains
2
Milk Protein Characterization by Mass Spectrometry
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All the chemicals used in the preparation of buffers and solutions were of analytical reagent grade. propan‐2‐ol (≥99.9%), methanol (≥99.9%), acetone (99.8%), acetic acid (HAc, glacial), formic acid (HFor, 99.0%), TFA (99.0%), hydrochloric acid (37% [v/v]), sinapinic acid (SA, ≥99.0%), sodium hydroxide (≥99.0%, pellets), potassium hydroxide (≥95.0%, pellets), sodium citrate dihydrate (≥99.0%), citric acid (99.5%), urea (99.0%–100.5%), DL‐DTT (97%), hydroxyethyl cellulose (HEC, relative molecular mass (Mr) of 250 000), hydroxypropyl cellulose (HPC, 100 000 Mr), β‐casein (βCN, ≥98%), β‐lactoglobulin (βLG, ≥90%), α‐casein (αCN, ≥70%), k‐casein (κCN, ≥70%), β‐lactoglobulin A (βLG‐A, ≥90%), and β‐lactoglobulin B (βLG‐B, ≥90%) from bovine milk were supplied by Merck (Darmstadt, Germany). ACN (LC‐MS grade) and water (LC‐MS grade) were provided by PanReac Applichem (Barcelona, Spain). Conventional ultra‐high temperature (UHT) skim (ten different brands) and semi‐skim milk (1 brand), as well as pasteurized semi‐skim milk that was supposed to have a higher content of β‐casein A2 (βCN‐A2) (one brand), were produced in Spain and acquired in a local market of Barcelona.
3
Assessing IgE Binding Capacity in WPI
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The capacity of both α-LA and β-LG in WPI to bind specific IgE was assessed by indirect competitive ELISA according to the method of Liu et al. [23 (link)] with slight modifications. α-Lactalbumin (L6010, purity 85%, Merck KGaA, Darmstadt, Germany) and β-lactoglobulin (L3908, purity 90%, Merck KGaA, Darmstadt, Germany) were dissolved in carbonate buffer solution (CBS, 0.05 mol/L, pH 9.6) at a concentration of 50 μg/mL and coated in ELISA plates, respectively, and serum diluted 20 times. Goat anti-human IgE antibody, biotin conjugate (Thermo Fisher Scientific, Shanghai, China) was dissolved in phosphate-buffered saline (PBS, 0.1 mol/L, pH 7.4) at a ratio of 1:2000. HRP-streptavidin (Solarbio, Beijing, China) was dissolved in PBS at a ratio of 1:2000.
OD values at 450 nm and 630 nm were measured on a microplate reader (Thermo Scientific, Massachusetts, USA). The inhibition rate was calculated according to the following formula:
ΔOD: The result of ELISA was the difference in the optical density of samples.
ΔOD0: The difference in optical density of noncompetitive.
OD values at 450 nm and 630 nm were measured on a microplate reader (Thermo Scientific, Massachusetts, USA). The inhibition rate was calculated according to the following formula:
ΔOD: The result of ELISA was the difference in the optical density of samples.
ΔOD0: The difference in optical density of noncompetitive.
4
Preparation of Cheddar-style Cheese Using Starter Cultures and Microbial Rennet
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The starter culture strains, Lactococcus lactis subsp. lactis XZ3303 and Lc. lactis subsp. cremoris QH27‐1, were obtained from the Inner Mongolia Agricultural University of China and stored in M17 medium containing 70% glycerine at −80°C. The two strains were activated by two consecutive transfers in M17 medium at 30°C and then propagated in milk for use in cheese making at a ratio of 1:1. The commercial rennet (CHY‐MAX Powder Extra NB, 3.17 × 105 Su (Soxhlet units)/g) used for making Cheddar‐style cheese in this study was purchased from Chr. Hansen, Inc. (Khusham, Denmark). The microbial rennet (1.09 × 105 Su/g) was isolated and partially purified from glutinous rice‐fermented wine that had been traditionally produced in southern China for centuries by using a rice starter called Jiuqu that mainly contains yeasts such as Saccharomyces cerevisiae (Zhao et al., 2015 ). The skim milk powder was purchased from Fonterra (New Zealand); α‐lactalbumin (≥85%) and β‐lactoglobulin (≥90%) powders were purchased from Sigma‐Aldrich (USA), which were reconstituted to the required concentration using distilled water. All chemical reagents used in this study were of analytical grade.
5
Protein Sample Preparation and Characterization
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Buffers were prepared using chemicals from Sigma Aldrich, UK, and the proteins were prepared according to the following protocols. β-lactoglobulin, thyroglobulin and BSA were obtained from Sigma Aldrich as lyophilised powders and made up to the desired concentrations by weighing and diluting in buffer. Concentrations were checked using a NanoDrop spectrophotometer (Thermo Fischer Scientific) before each measurement. Full-length human αB-crystallin (αBC), core-domain αBC S135W (residues 68-153 with a point mutation) and core-domain Hsp27 (residues 84-176) were expressed and purified as previously described. 20 Where reduction of core-domain Hsp27 was required, a solution of dithiothreitol (DTT) stock solution was prepared in water and added to the core-domain Hsp27 samples at a molar ratio of 1:10 protein:DTT.
6
Protein Purification and Characterization
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Bovine serum albumin (BSA), ovalbumin from egg white, -lactoglobulin from bovine milk ( -lac), lysozyme from chicken egg white, and the N -Acetyl-l -lysine (NAK) were all purchased from
Sigma-Aldrich. The calmodulin sample was kindly provided by Professor Sara Linse
(Department of Biochemistry and Structural Biology, Lund University).
Sigma-Aldrich. The calmodulin sample was kindly provided by Professor Sara Linse
(Department of Biochemistry and Structural Biology, Lund University).
7
Microfluidic Droplet Generation of Protein Solutions
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The flow rates within the channels were controlled using neMESYS syringe pumps (Cetoni, Korbussen, Germany). For water-in-oil droplets, protein solution was used as the dispersed phase, while fluorinated oil (Fluorinert FC-40, Sigma Aldrich) containing 2% w/w fluorosurfactant (RAN Biotechnologies) was used as the continuous phase. Protein stock solutions of 4, 50, and 50 mg/ml were prepared for reconstituted silk fibroin (purification process mentioned below), β-lactoglobulin (Sigma-Aldrich), and lysozyme (Sigma-Aldrich), respectively. Note that β-lactoglobulin and lysozyme were prepared in a pH 1.6 solution, which was adjusted using HCl. In addition, Mikrotron high-speed cameras were used for imaging.
8
Protein Sample Preparation Conditions
9
CCS Calculation and Calibration
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CCSs (Ω) of models from the PDB (trimmed in PyMOL as described in the Supplementary Materials) were calculated by the projection approximation method using IMPACT (60 (link)), accounting for N2 as the collision gas. To calculate CCSs of FLNCd18–21, native mass spectra of β-lactoglobulin and avidin (Sigma) were collected under identical drift cell conditions (as described for titration experiments, with the mobility wave height lowered to 5.5 V) and used to construct a calibration in PULSAR, which had R2 > 0.96, using a power law linear fit.
10
Chemiluminescent Protein Detection Assay
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Bovine serum albumin (BSA), ovalbumin from egg white, β-lactoglobulin from bovine milk (β-lac), lysozyme from chicken egg white and the Nα-Acetyl-L-lysine (NAK) were all purchased from Sigma Aldrich. The calmodulin sample was kindly provided by Prof. Sara Linse (Department of Biochemistry and Structural Biology, Lund University). In parallel, a reaction between hydrogen peroxide and bis-(2,4,6-trichlorophenyl) oxalate (TCPO) yields a high energy four-membered ring (yellow box), which transfers its energy to the benzoisoindole. The relaxation of benzoisoindole down to its ground state releases a photon that can be captured as a chemiluminescent (CL) signal (blue box). NDA is a fluorogenic molecule, rendering the assay free of a requirement to remove unbound probe molecules.
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