Experimental procedures were previously described (23 (link)). Briefly, wild type MVA (B. Verrier, Laboratoire de Biologie tissulaire et d’ingénierie thérapeutique, Institute of Biology and Chemistry of Proteins, Lyon, France) was used to coat 96-well MaxiSorp microplates (Nunc) at 105 PFU/well in coating buffer (200 mM NaHCO3, 80 mM Na2CO3, pH 9.5) overnight at 4˚C. Wells were washed five times with wash buffer (PBS, 0.1% Tween 20, 10 mM EDTA) and blocked for 1 h at room temperature (RT) with 3% w/v BSA (Sigma). Plates were washed five times and incubated with two- or four-fold serial dilutions of macaque serum diluted in 1% w/v BSA in PBS for 2 h at RT, starting at 1:50 for IgG and 1:25 for IgM. Plates were then washed five times and peroxidase-conjugated goat anti-monkey H+L chain IgG (1:2,500) or IgM (1:1,000) (AbD Serotec) in 1% BSA (w/v) PBS was added and the plates incubated for 1 h at RT. Plates were washed five times, and 100 mL 3,3’,5,5’-tetramethylbenzidine (Thermo Scientific) added and the plates incubated for 30 min at RT in the dark. The reaction was stopped by adding 2N H2SO4. Absorbance was measured at 450 nm using a spectrophotometer (Multiskan FC, Thermo Scientific) and the data analyzed using SoftMax Pro software (version 4.6; Molecular Devices). Ab titers were calculated as described (23 (link)).
Bovine serum albumin (bsa)
Manufactured by Bio-Rad
Sourced in United States, Germany, United Kingdom, Canada, Italy, France, Japan, China
Bovine serum albumin is a common laboratory reagent used as a protein standard and stabilizer in various biological assays and experiments. It is derived from the serum of cattle and serves as a source of protein for cell culture media and other applications.
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Lab products found in correlation
Protein assay, by Bio-Rad (23 mentions)
Bradford assay, by Bio-Rad (18 mentions)
Bradford reagent, by Bio-Rad (11 mentions)
Protein assay kit, by Bio-Rad (10 mentions)
Dc protein assay kit, by Bio-Rad (9 mentions)
Bovine serum albumin (bsa), by Merck Group (7 mentions)
Protease inhibitor cocktail, by Merck Group (7 mentions)
Triton x 100, by Merck Group (6 mentions)
Nitrocellulose membrane, by Bio-Rad (6 mentions)
Bradford method, by Bio-Rad (6 mentions)
Bradford protein assay, by Bio-Rad (5 mentions)
Lysis buffer, by Thermo Fisher Scientific (4 mentions)
Ecl reagent, by GE Healthcare (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Dc protein assay, by Bio-Rad (3 mentions)
Non fat dry milk, by Bio-Rad (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Protein assay reagent, by Bio-Rad (3 mentions)
Chamber slide, by SPL Life Sciences (3 mentions)
299 protocols using bovine serum albumin (bsa)
1
ELISA Protocol for Detecting Macaque Antibodies
2
Immunophenotyping of Dissociated Cells
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Cells were dissociated with Accutase, fixed in 1% PFA for 15 min at room temperature, and then washed with 0.5% bovine serum albumin (BSA) (Bio-Rad) plus 0.1% Triton X-100 three times. Cells were stained with primary and secondary antibodies diluted in 0.5% BSA plus 0.1% Triton X-100, as previously described (31 (link)). Data were collected on a FACSCalibur flow cytometer (Becton Dickinson) and analyzed using FlowJo. Corresponding isotype antibodies were used as FACS (fluorescence-activated cell sorting) gating control. Details about antibody source and usage are provided in table S2.
3
SDS-PAGE Protein Separation and Visualization
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Total egg extract and proteolytic products were mixed with an equal volume of 2x sample buffer (62 mM Tris, 50 mM DTT, 0.2% SDS, 10% glycerol, and 0.01% bromophenol blue). Proteins were resolved by SDS-PAGE using a 12% gel [35 (link)] and visualised by staining with 0.2% Coomassie Brilliant Blue R250 (w/v) dissolved in ethanol, acetic acid, and water (45:10:45, v/v/v) (Bio-Rad Laboratories, Brazil), and the molecular masses were estimated using the following protein standards: phosphorylase B (101.4 kDa), bovine serum albumin (87.5 kDa), ovalbumin (52.7 kDa), carbonic anhydrase (35.8 kDa), soybean trypsin inhibitor (22.8 kDa), and lysozyme (18.8 kDa) (Bio-Rad Laboratories, USA).
Total ovary extracts were resolved using 8% SDS-PAGE [35 (link)] by the same process described above, and molecular masses were estimated using the following protein standards: myosin (202.8 kDa), β-galactosidase (115.5 kDa), bovine serum albumin (98.2 kDa), ovalbumin (51.4 kDa) (Bio-Rad Laboratories, USA).
Total ovary extracts were resolved using 8% SDS-PAGE [35 (link)] by the same process described above, and molecular masses were estimated using the following protein standards: myosin (202.8 kDa), β-galactosidase (115.5 kDa), bovine serum albumin (98.2 kDa), ovalbumin (51.4 kDa) (Bio-Rad Laboratories, USA).
4
Fibroblastic Differentiation of ADSCs
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The culture techniques were performed as described in our previous study [17] . Briefly, adipose tissue (approximately 8–10 g) was collected from the inguinal area, cut into small pieces, and then digested at 37°C for 40 minutes using 0.2% collagenase type I (Sigma-Aldrich, St. Louis, USA) and 2% bovine serum albumin (AbD Serotec, Kidlington, UK). Enzyme activity was neutralized with DMEM containing 10% FBS. The samples were filtered through a 100-μm nylon mesh to remove cellular debris and were incubated overnight at 37°C with 5% CO2 in DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. The medium was changed every 1 to 2 days. The subculture and identification techniques were described in our previous study [17] .
The fibroblastic differentiation-inducing medium consisted of DMEM supplemented with 100 ng/ml connective tissue growth factor (CTGF; Biovendor, Brno, Czech Republic) and 50 μg/ml ascorbic acid (Sigma-Aldrich, St. Louis, USA) with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. ADSCs cultured in normal DMEM were used as a control. The medium was changed twice each day. On days 7, 14 and 21, the dADSCs were collected for observation and cell immunofluorescence studies.
The fibroblastic differentiation-inducing medium consisted of DMEM supplemented with 100 ng/ml connective tissue growth factor (CTGF; Biovendor, Brno, Czech Republic) and 50 μg/ml ascorbic acid (Sigma-Aldrich, St. Louis, USA) with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. ADSCs cultured in normal DMEM were used as a control. The medium was changed twice each day. On days 7, 14 and 21, the dADSCs were collected for observation and cell immunofluorescence studies.
5
Histological Analysis of Immune Organs
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For histological analysis, tissues and organs, including the spleen, lymph nodes, thymus, liver, and femur/tibia, were fixed in 4% formalin during 24 h. Bone-containing tissues were decalcified using Osteosoft (Merck Laboratories). Specimens were embedded in paraffin, and 4-mM serial sections were cut and stained with hematoxylin and eosin following standard procedures. After dewaxing, rehydration, and antigen retrieval using sodium citrate, slides were blocked with 2% normal goat serum (Vector Laboratories) and 2% bovine serum albumin in 0.05% phosphate-buffered saline, Tween20 prior to overnight staining with F4/80 (MCA497GA; AbD Serotec), and asparaginase (ab21013; Abcam) antibodies. Appropriate biotinylated antibodies (Vector Laboratories) were used as secondary antibody, and staining was revealed by incubation with metal-enhanced diaminobenzidine in stable peroxide substrate buffer (Thermo Fisher Scientific Inc.). After being counterstained with hematoxylin (1:3), slides were dehydrated and coverslipped using entellan (Merck Laboratories). Images were captured using a VisionTek digital microscope (Sakura Finetek).
6
Western Blot Analysis of Autophagy Markers
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Total proteins were extracted using RIPA buffer (Beyotime, Shanghai, China). The protein samples were loaded on SDS-PAGE gel, separated by electrophoresis, and then transferred on a PVDF membrane (Millipore) that was further incubated with primary antibodies for LC-3B, p62, and GAPDH (Cell Signaling Technology, Danvers, MA, USA) after blocking with 5% BSA (Bio-Rad). Next, the bands were visualized by a gel imager with enhanced ECL reagents (Advansta, San Jose, CA, USA), and images were obtained by Electrophoresis Image Analysis System (Peiqing Science & Technology, Shanghai, China). The protein gray densities of internal control and target were quantified by Image J software.
7
Quantitative Western Blot Analysis
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Homogenized brain cell proteins separated by 13.5% homologous SDS-PAGE were electrophoretically transferred to a polyvinylidene difluoride (PVDF, MSI, Westborough, US) membrane. The membranes were blocked with 5% BSA (Bio-Rad) in PBS for 30 min at 37°C, and then incubated with diluted 1° Ab at 37°C for 1 h. After washing, membranes were incubated with HRP-conjugated 2° Ab at 37°C for 0.5 h. Protein expression was detected using an ECL-Plus Western Blot Detection system (GE Healthcare UK Ltd.) according to the manufacturer's instructions. All experiments were performed at least thrice. Graphical analysis of band density was performed using ImageJ software (version 1.41o) (National Institutes of Health, Bethesda, MD, USA) (http://rsb.info.nih.gov/ij/). We used anti-YM-1, and Iba-1 antibodies purchased from R & D Systems (Minneapolis, MN), and the BD Biosciences (Billerica, MA) company.
8
Conjugation of ZW800 Fluorophores
9
Protein Quantification: Bradford Assay Optimization
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Cells were collected in PBS by scraping from culture flasks and then washed twice with cold PBS. The slurry was centrifuged and lysis buffer consisting of 1.0 ml NP40 supplemented with protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) was added to the pellet, then incubated for 30 minutes on ice with vortexing every 10 minutes. The cell extract was then transferred to a microfuge tube and centrifuged at 13,000 rpm for 10 minutes at 4 °C. Clear lysates were then aliquoted into clean microfuge tubes and stored at 4 °C.
To determine total protein, a Bradford Assay was carried out. However, we modified the manufacturer's instructions by reducing the volume of protein standard, containing BSA, (obtained from Biorad) from 50 μl to 5 μl per well. PC-12 and LNCaP lysates were added and dH2O served as a control. To compensate for the decreased volume, 250 μl of Bradford reagent was added to each well with a multi-channel pipettor. Standards, control and lysates were run in triplicate. The plates were placed on a shaker for 10 minutes then read at an absorbance of 595 nm. SeeFig. 2 .Fig. 2 ![]()
To determine total protein, a Bradford Assay was carried out. However, we modified the manufacturer's instructions by reducing the volume of protein standard, containing BSA, (obtained from Biorad) from 50 μl to 5 μl per well. PC-12 and LNCaP lysates were added and dH2O served as a control. To compensate for the decreased volume, 250 μl of Bradford reagent was added to each well with a multi-channel pipettor. Standards, control and lysates were run in triplicate. The plates were placed on a shaker for 10 minutes then read at an absorbance of 595 nm. See
Protein totals in each lysate via Bradford Assay.
10
Xanthohumol Modulates NF-κB Signaling in HepG2 Cells
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HepG2 cells were seeded at a density of 1×106 cells/culture dish. After xanthohumol treatment, cells were washed with cold PBS and lysed with cold RIPA buffer containing protease inhibitors. Protein concentrations were measured using BSA (Bio-Rad Laboratories, Hercules, CA, USA). Next, 40 µg protein was separated with 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.2 µm nitrocellulose membrane blocking with 1% BSA in PBS-T. Each membrane was incubated with a specific primary antibody NF-κB (1:1,000), p53, PARP, XIAP, AIF, Bax, cytochrome c and β-actin at 4°C overnight. After three washes in 1% BSA in PBS-T, each membrane was incubated with the appropriate secondary antibody at room temperature for 2 h and visualized using an ECL Advanced Western blot detection kit (Thermo Fisher Scientif, Waltham, MA, USA).
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