To elucidate which matrix components are most likely explaining different degrees of protection was observed in the presence of the three matrices, a spiking approach with protein, and fat was applied to the matrix lowest in protein (DM) and to the one lowest in fat (SM), respectively. To investigate a potential protective role of protein content, PT was digested using the sequential digestion protocol in DM (1.75% protein) and in the same matrix spiked with 1.25% bovine serum albumin (BSA; Roche Diagnostics GmbH, Mannheim, Germany) to mimic the protein content of SM (3% protein) or with 10.75% BSA to mimic the protein content of the CB matrix (12.5% protein). To investigate a potential protective role of fat content, PT was digested in SM (1.8% fat) and in the same matrix spiked with 8.2% fat (Choc chick raw organic cacao butter/99.8% fat; Holland & Barrett, Warwickshire, UK) to mimic the fat content of DM (10% fat) or with 44.2% fat to mimic the fat content of the CB (46% fat).
Bovine serum albumin (bsa)
Manufactured by Roche
Sourced in Germany, Switzerland, United States, China, Canada, France, United Kingdom, Italy, Hong Kong, Belgium
BSA (Bovine Serum Albumin) is a laboratory product that serves as a protein standard. It is commonly used in various assays and experiments to quantify the concentration of other proteins.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (23 mentions)
Triton x 100, by Merck Group (13 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (9 mentions)
Pvdf membrane, by Merck Group (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Epidermal growth factor (egf), by Merck Group (6 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (6 mentions)
Lsm 780 confocal microscope, by Zeiss (6 mentions)
Bca protein assay, by Thermo Fisher Scientific (5 mentions)
Rotiszint eco plus, by Carl Roth (5 mentions)
Complete protease inhibitor mixture, by Roche (5 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Tween 20, by Merck Group (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Hoechst 33342, by Merck Group (4 mentions)
Cholera toxin, by Merck Group (4 mentions)
Paraformaldehyde (pfa), by Merck Group (4 mentions)
395 protocols using bovine serum albumin (bsa)
1
Investigating Protein and Fat Influence on Digestion
2
Western Blot Analysis of Cleaved Caspase-3
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Western blotting was performed as described previously.18 (link) Tissue samples were manually homogenized with a glass homogenizer, and whole tissue extracts were prepared with radioimmunoprecipitation assay buffer (RIPA) whole cell lysis buffer supplemented with 1× phosphatase and protease inhibitors (Halt, Thermo Fisher Scientific, Waltham, MA). The supernatant was collected after centrifugation at 10 000 RPM at 4°C for 15 minutes. Prior to loading on a 12% polyacrylamide gel, whole cell extracts were balanced by spectrophotometry using the Bradford assay to ensure equal loading. Once separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, proteins were electrotransferred onto a nitrocellulose membrane. Membranes were blocked for 30 minutes using 5% bovine serum albumin (Roche, Indianapolis, IN) dissolved in Tris-buffered saline infused with 0.035% Tween 20 (TBST) and then incubated with the primary antibody diluted in TBST containing 5% bovine serum albumin at 4°C overnight. Rabbit anti-cleaved caspase 3 (c-caspase 3, 1:1000) from Cell Signaling Technology (Danvers, MA) was used as the primary antibody.
3
Immunostaining of Drosophila Antennae
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White puparia were aged for the appropriate period of time at 25 o C. The anterior puparium case was opened up and removed to uncover the pupal head region and the whole pupa was fixed in 3.7% formaldehyde for 30 minutes, followed by washing in PBT (0.3% Triton X-100). Subsequently, the antennae were dissected off, collected in a 1.5 ml tube and blocked overnight in 3% BSA (Roche) before incubating in primary antibody (3% BSA in PBT) at 4 o C for 2 days. After washing in PBT, antennae were incubated in secondary antibodies at 4 o C overnight. They were mounted in a 2.5% n-Propyl gallate/glycerol mix. The following primary antibodies were used: Goat anti-GFP (1:500:
4
Quantitative Western Blot Analysis
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Cells (4 × 105 per point) were lysed in 150 µL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, pH 8.0) supplemented with protease and phosphatase inhibitors (Roche Diagnostics, Meylan, France) for 3 h at 4 °C. Lysates were then clarified by centrifugation and protein content of the supernatants was estimated using micro-BCA protein assay kit (Thermo Fisher Scientific). Samples corresponding to 20 µg of proteins were mixed with Laemmli buffer and boiled for 10 min, after which proteins were separated by SDS-PAGE and transferred onto nitrocellulose (Amersham, Uppsala, Sweden). The membrane was blocked for 1 h in 20 mM Tris buffer (TBS) with 0.05% (v/v) Tween-20 and 5% (w/v) BSA (Roche), and then probed with primary antibodies overnight at 4 °C in TBS supplemented with 3% (w/v) BSA. After washing, HRP-conjugated secondary antibodies (1:10,000) were added for 1 h and immunoreactive proteins were detected using ECL prime Western blotting detection (GE Healthcare). Quantification of immunostaining intensity was performed by using Image J software.
5
Immunocytochemical Staining of Vascular and Cancer Cells
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To visualize the vascular structures and cancer cells, immunocytochemical staining was performed. Co-cultures were fixed with 70% ethanol at day 7. After fixation, the cells were permeabilized with 0.5% Triton-X100 (MP Biochemicals, Solon, OH, USA) and non-specific binding sites were blocked with 10% BSA (Roche Diagnostics). Primary and secondary antibodies were applied in 1% BSA (Roche). Primary antibodies utilized were rabbit anti-human VWF IgG (1:100, F3520, Sigma and A0082, DAKO), basement membrane marker mouse anti-human collagen IV (anti-COLIV, clone COL-94, 1:500, C1926, Sigma), rabbit anti-human ALDH1A1 (1:250, ab52492, Abcam, Cambridge, UK), rabbit anti-human CD44 (1 µg/mL, ab157107, Abcam), mouse anti-human CK19 monoclonal antibody (2 µg/mL, RCK108, Thermo fisher scientific), secondary antibodies anti-rabbit IgG A568 (1:400, ab175471, Abcam), and anti-mouse IgG fluorescein isothiocyanate (FITC, 1:100, F4143, Sigma). A drop of Fluoroshield™ with DAPI histology mounting medium (Sigma) was added into the emptied wells to stain the nuclei and mount the cultures.
6
Western Blot Analysis of MDA-MB-231 Cells
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MDA-MB-231 cells (4×105 per point) were lysed in 150 μL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, pH 8.0) supplemented with a mixture of protease and phosphatase inhibitors (Roche Diagnostics, Meylan, France) for 3 h at 4°C. Lysates were clarified by centrifugation at 10,000 g for 30 min at 4°C. Protein content of the supernatants was estimated using micro-BCA protein assay kit (Thermo Fisher Scientific). Samples corresponding to twenty micrograms of proteins were mixed with Laemmli buffer and boiled for 10 min. Proteins were then separated by SDS-PAGE and transferred onto nitrocellulose membrane (Amersham, Uppsala, Sweden). The membrane was blocked for 1 h at room temperature in 20 mM Tris-HCl, pH 7.6, 150 mM NaCl (TBS) with 0.05% (v/v) Tween-20 and 5% (w/v) BSA (Roche), and then probed with primary antibodies (1/2000) overnight in TBS supplemented with 5% (w/v) BSA. After washing, HRP-conjugated secondary antibodies (1/10,000) were added for 1 h at room temperature and immunoreactive proteins were detected using ECL prime Western blotting detection (GE Healthcare). Quantification of immunostaining intensity was performed by using Image J software.
7
TGF-β Signaling Pathway Modulation
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RCC cells were grown in serum-free medium supplemented with 0.1% BSA (Roche) and 2 mM glutamine (MilliporeSigma) for 24 hours prior to treatment with either TGF-β (1 ng/mL, Calbiochem) or 10 µM TGF-β inhibitors (SB431542, Sigma-Aldrich; LY2109761 and LY364947, Selleckchem) for the indicated times. HK2 cells were cultured with 3% FBS prior to exposure with serum-free medium supplemented with 0.1% BSA (Roche) and 2 mM glutamine for 24 hours, followed by treatment with TGF-β (1 ng/mL).
8
Immunofluorescence Quantification of SV2A Puncta
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Immunofluorescence staining was performed as previously described (Nicholson-Fish et al., 2015) . Briefly, hippocampal neurons were fixed with 4 % paraformaldehyde (Sigma Aldrich, 47608) in PBS for 15 min. Excess paraformaldehyde was quenched with 50 mM NH4Cl in PBS. Cells were then permeabilized in 1 % bovine serum albumin (BSA; Roche Diagnostics GmbH, Germany, 10735078001) in PBS-Triton 0.1 % solution for 5 min and blocked in 1 % BSA in PBS at room temperature for 1 h. After blocking, cells were incubated in rabbit anti-SV2A (1:200 dilution) for 1 h, after which the cultures were washed with PBS and incubated with fluorescently conjugated secondary antibodies (anti-rabbit Alexa Fluor 488; 1:1000 dilution) for 1 hr. The coverslips were mounted on slides for imaging with FluorSave (Millipore, Darmstadt, Germany, 345789). SV2A puncta were visualised at 500 nm band pass excitation with a 515 nm dichroic filter and a long-pass >520 nm emission filter on a Zeiss Axio Observer D1 inverted epifluorescence microscope (Cambridge, UK). Images were captured using an AxioCam 506 mono camera (Zeiss) with a Zeiss EC Plan Neofluar 40x/1.30 oil immersion objective. SV2A puncta in each image were quantified using the Analyze Particles plugin of Image J to select and count particles of 0.23-3.18 µm 2 .
9
Immunocytochemical Analysis of Neural Stem Cells
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NSCs were fixed in 4% PFA for 1 h at 4°C, followed by washing in PBS. The NSCs were incubated with mouse anti-tubulin III (Tuj1, 1 : 400; Covance, Princeton, NJ, USA) and rabbit anti-glial fibrillary acidic protein (GFAP, 1 : 800; DakoCytomation, Glostrup, Denmark) diluted in PBS containing 0.3% Triton X-100 and 0.5% bovine serum albumin (BSA; Boehringer Mannheim, Mannheim, Germany) overnight in a humid chamber at 4°C. The cells were then rinsed with PBS and incubated with an appropriate secondary FITC-conjugated antibody for 1 h at RT (1 : 200; Alexa, Invitrogen, Carlsbad, CA, USA). Cell nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich) (1 mg/ml) for 5 min. After rinsing with PBS, the coverslips were mounted onto slides with VECTASHIELD mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA). The cells were examined using fluorescence microscope (Nikon Eclipse Ti-S, BergmanLabora AB, Danderyd, Sweden) and images were captured using Nikon camera (Nikon Digital Sight DS-Qi1MC, BergmanLabora AB). All experiments were performed in triplicates and repeated three times. Semiquantitative analyses were performed by counting at least 100 cells per coverslip in triplicates.
10
Quantification of Collagen IV α5 in Cell Lysates
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Bovine GBM was purified as described previously28 (link) and coated on plates at 20 μg/ml in phosphate-buffered saline, pH 7.3, overnight at 4 °C (Nunc immunoplates). Plates were then blocked with 1% bovine serum albumin (Boehringer Mannheim, Mannheim, Germany) in phosphate-buffered saline for 2 hours at room temperature. Concentrations of GBM were determined in an inhibition assay performed as follows. Twenty-five microliters of intracellular lysate prepared from the supernates after microfugation, from 5 freeze-thaws in phosphate-buffered saline, or 25 μl of the extracellular culture supernate, all corrected for cell numbers, were preincubated with 25 μl of 1:50 dilution rabbit anticollagen IV α5 antibody in phosphate-buffered saline/TWEEN (Sigma-Aldrich) at room temperature for 30 minutes and then added to GBM-coated wells in duplicate for 1 hour at room temperature. The plates were then incubated with a 1:1000 dilution of alkaline phosphatase–conjugated goat anti-rabbit IgG (Sigma-Aldrich) for 30 minutes and p-nitrophenylphosphate (Sigma-Aldrich) for 15 minutes at room temperature. The absorbance was measured at 405 nm (Thermo Scientific Fisher plate reader), and the concentrations were determined from a standard curve of serial dilutions of GBM with final concentrations of 0.03 to 30 μg/ml.
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