The standards were prepared by dissolving 32 mg BSA (Biotechnology, Grade Amresco, 0332) in 40 mL 1x-PBS solution (0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4, at 25°C) (Sigma, P4417), and further diluting the solution to 80 ng/mL. In Eppendorf tubes, a twofold dilution series of 80 ng/mL to 5 ng/mL BSA were prepared. A PBS solution without BSA was used as the 0 ng/mL standard.
Bovine serum albumin (bsa)
Manufactured by Eppendorf
Sourced in Switzerland
The BSA is a laboratory instrument that performs biomass determination. It utilizes spectroscopic analysis to provide accurate measurements of protein concentration in solution.
Automatically generated - may contain errors
4 protocols using bovine serum albumin (bsa)
1
Preparation of BSA Dilution Series
2
BSA-YCl3 Interaction Phase Diagram
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The materials used in this study were purchased from Sigma Aldrich, namely BSA with a purity of ≥98% (product No. A7906) and YCl3 with a purity of 99.99 % (product No. 451363). SA is one of the most abundant blood proteins in mammals and has a net negative charge of −10e at neutral pH46 (link). The protein solutions were prepared with degassed Milli-Q water at cp of 5, 20, and 50 mg/ml and T ranging from 10 to 40 °C. cs was varied from 0 to 60 mM. While in this study we focus on BSA, we emphasize that the effects are expected to be rather general, as indicated in tests with, e.g. BLG.
The temperature-dependent phase diagram shown in Fig.1(a) was generated with the Thermostat C of Eppendorf for a stable temperature control. The phase transitions were determined by eye based on onset of turbidity in Fig. S2 . Typical aggregation sizes and its formation was part of a previous study by Soraruf et al.47 (link). For the lowest investigated protein concentration of cp = 5 mg/ml, c* and c** had to be determined via UV-Vis spectroscopy measurements with the Cary 50 UV-visible spectrometer of Varian Technologies since the turbidity detection by eye was not sufficiently precise, see Fig. S2 45 . The spectrometer was also used to perform absorbance measurement to determine the protein stock solution concentration via the Lambert-Beer law.
The temperature-dependent phase diagram shown in Fig.
3
Growth Factor Release from Collagen Matrices
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In vitro analysis of growth factor release from each of the collagen matrices was performed by incubating the matrices in 3 mL PBS, pH 7.2 containing 0.1% bovine serum albumin (BSA; Sigma, Buchs, Switzerland) and 1% antibiotics/antimycotics (AA; ThermoFisher Scientific, Basel, Switzerland) solution at 37 °C with gentle shaking at 70 rpm for 13 days. Protein quantification by ELISA was performed 0.5, 1, 2, 3, and 6 h, and 1, 3, 5, 7, 9, 11, and 13 days after growth factor loading. At each time point, the 3 mL of supernatant were collected, frozen in low protein-binding tubes (Eppendorf, Basel, Switzerland), and replaced with 3 mL of fresh PBS/BSA/AA solution. The amount of released protein was calculated as percent of the adsorbed protein. Earlier studies identified TGF-β1 in EMD by immunoassays [57 (link)] and had shown that TGF-β-like activity can be passively released from EMD-coated collagen products [26 (link)], which allowed us to test the release of TGF-β1 from EMD-coated collagen matrices. Three independent experiments with three replicates were performed for each experimental group.
4
Protein Aggregation Assay Protocol
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Microscope
cover slides (Chance Propper) were washed once with methanol, followed
by two washes with Milli-Q water. To reduce protein binding, the cover
slides were incubated for 30 min in the fridge with 0.2 μg/μL
bovine serum albumin (BSA) (Thermo Fisher Scientific). Excess BSA
was removed by three washes with 50 mM HEPES buffer containing 150
mM NaCl at pH 7.4. The K562 lysate was treated with 10 μM MTX
or an equal amount of DMSO for 15 min at RT. A sample containing 1
μg of protein was divided into five samples of 200 ng each,
which were pipetted onto the cover slides. ZnCl2 was added
(starting at 100 μM, steps of 100 μM), and samples were
carefully pipetted for mixing. After 10 min, kosmotropic ions were
quenched by adding a two times molar excess of Na2HPO4. After 10 min, samples were pooled into low bind tubes (Eppendorf),
which were coated with BSA the same way as the cover slides. Samples
were centrifuged for 20 min at 20,000g at 4 °C
to remove aggregated proteins. Samples were reduced with 5 mM dithiothreitol
(DTT) for 1 h at RT, followed by alkylation with 15 mM IAA for another
hour in the dark. Then, 20 ng of trypsin (Promega) was added, and
samples were incubated overnight at RT. Peptide mixtures were acidified
with TFA to pH < 3 and cleaned by StageTips. Samples were dried
in a SpeedVac (Genevac) and stored at −80 °C.
cover slides (Chance Propper) were washed once with methanol, followed
by two washes with Milli-Q water. To reduce protein binding, the cover
slides were incubated for 30 min in the fridge with 0.2 μg/μL
bovine serum albumin (BSA) (Thermo Fisher Scientific). Excess BSA
was removed by three washes with 50 mM HEPES buffer containing 150
mM NaCl at pH 7.4. The K562 lysate was treated with 10 μM MTX
or an equal amount of DMSO for 15 min at RT. A sample containing 1
μg of protein was divided into five samples of 200 ng each,
which were pipetted onto the cover slides. ZnCl2 was added
(starting at 100 μM, steps of 100 μM), and samples were
carefully pipetted for mixing. After 10 min, kosmotropic ions were
quenched by adding a two times molar excess of Na2HPO4. After 10 min, samples were pooled into low bind tubes (Eppendorf),
which were coated with BSA the same way as the cover slides. Samples
were centrifuged for 20 min at 20,000g at 4 °C
to remove aggregated proteins. Samples were reduced with 5 mM dithiothreitol
(DTT) for 1 h at RT, followed by alkylation with 15 mM IAA for another
hour in the dark. Then, 20 ng of trypsin (Promega) was added, and
samples were incubated overnight at RT. Peptide mixtures were acidified
with TFA to pH < 3 and cleaned by StageTips. Samples were dried
in a SpeedVac (Genevac) and stored at −80 °C.
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