China
Africa
Explore chapters and articles related to this topic
*
Published in Chad A. Mirkin, Spherical Nucleic Acids, 2020
Hui Li, Bohan Zhang, Xueguang Lu, Xuyu Tan, Fei Jia, Yue Xiao, Zehong Cheng, Yang Li, Dagoberto O. Silva, Henri S. Schrekker, Ke Zhang, Chad A. Mirkin
SKOV3 cells were plated in a 24-well plate at a density of 1.0 × 105 cells per well and cultured for 24 h. Then medium was replaced with serum-free DMEM immediately before treatment with free DNA (200 nM), POSS SNA (500 nM), C60 SNA (500 nM, 1000 nM), and Lipofectamine-complexed DNA (200 nM, following manufacturer suggested protocol). After 15 h, the medium was replaced with fresh, full growth medium, and cells were cultured for another 48 h. Whole cell lysates were prepared in 75 μL of radioimmunoprecipitation assay (RIPA) Cell Lysis Buffer (Sangon Biotech). Protein concentrations were determined using a bicinchoninic acid assay (BCA) Protein Assay Kit (Sangon Biotech). Equal amounts (25 μg) of protein samples were fractionated by 4 to 20% precast gradient gels (Beyotime), transferred to polyvinylidene difluoride (PVDF) membrane, and blocked with 5% nonfat milk in tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature (Bio-Rad). Proteins were analyzed by Western blotting with rabbit primary antibodies against HER2 (1000:1) (Cell Signaling), rabbit primary antibodies against GAPDH (1000:1) (Sangon Biotech), and HRP-conjugated goat anti-rabbit IgG secondary antibodies (2000:1) (Sangon Biotech) using an ECL Western blotting substrate (Beyotime).
In Situ Nanotechnology-Derived Sensors for Ensuring Implant Success
Published in Šeila Selimovic, Nanopatterning and Nanoscale Devices for Biological Applications, 2017
Sirinrath Sirivisoot, Thomas J. Webster
To study osteoblast differentiation between non-Ca- and Ca-depositing cells, osteo-blasts were seeded at a density of 40,000 cells/cm2 in DMEM supplemented with 10% FBS, 1% P/S, 50 nM β-glycerophosphate (Sigma), and 50 μg/mL ascorbic acid (Sigma) under standard cell culture conditions (a humidified 5% CO2 and 95% air environment at 37°C) for 7, 14, and 21 days. The cell media were changed every other day. At the end of each time point, an alkaline/acid phosphatase assay kit (Upstate) was used to determine the concentration of alkaline phosphatase in cell lysates. The cell lysates were prepared by first rinsing all samples three times with Tris-buffered saline (TBS; 42 mM Tris-HCl, 8 mM Tris base, and 0.15 M NaCl; a pH of 7.4; Sigma-Aldrich) and then subjecting the cells to three freeze–thaw cycles using distilled water. A Ca quantification kit (Sigma) was used to determine the amount of Ca deposited by the osteoblasts seeded on each sample. An acidic super-natant solution for a Ca deposition assay was prepared by incubating all the samples with 0.6 N HCl (Sigma) for 24 h. The light absorbance was measured by a spectrophotometer (SpectraMAX 340PC384; Molecular Devices) at 650 nm for alkaline phosphatase activity and 570 nm for Ca deposition. Long-term cytocompatible testing was conducted in triplicate and was repeated three different times. The numerical data were analyzed using standard Student’s t-test.
Thin-Layer Chromatography in Bacteriology
Published in Bernard Fried, Joseph Sherma, Practical Thin-Layer Chromatography, 2017
The choice of blocking reagent is important. Without a blocking agent, background staining is usually quite dark, making the results unacceptable. One percent gelatin in 50 mM tris-buffered saline (TBS) is often used, although blocking with typically 1 to 2% or less of albumin, serum from various sources, powdered milk, Tween 20, and trimethyl urea have been used with success. The ideal blocking protocol must be determined empirically for each system. The choice of BSA over gelatin has been shown to diminish the binding of bacteria to some glycolipids in at least one study.40 However, there is some doubt as to whether the observed binding in the absence of BSA was physiologically relevant or artifactual. Blocking with gelatin should be performed at 37°C for approximately 1 h or longer if required. Excessive blocking may result in background staining of the plate. Again, the ideal blocking scheme must be determined for each system.
Toxic effects of Aroclor 1254 on rat liver and modifying roles of selenium
Published in International Journal of Environmental Health Research, 2023
Aylin Balcı Özyurt, Pınar Erkekoğlu, Naciye Dilara Zeybek, Ali Aşcı, Ünzile Yaman, Ofcan Oflaz, Murat Kızılgün, Evin İşcan, Tuğçe Batur, Mehmet Öztürk, Belma Koçer-Gümüşel
Throughout the experiments, HepG2 cells were used as positive controls. β-actin was used as a loading control. Total cell lysates for HepG2 cells were obtained with lysis buffer. “BCA protein assay kit” was used to determine the amount of protein in the supernatants. 30 µg total protein was used for the determination of each protein. After protein denaturation, Tris-glycine electrophoresis buffer was added into the electrophoresis tank (BioRad, Hercules, CA) and samples were added into the gels. To monitor protein migration, Reagents (SM0441 and SM06714, 4 μl each) were loaded and run until bands were observed appropriately. Gels were removed from the glass apparatus and placed in a cassette. By passing current through transfer tank, proteins in the gel were transferred to the membrane in the cassette. This membrane was removed and blocked in 5% BSA. Membranes from blocking solution were treated with primary antibody at optimized dilutions. Anti-p53 sc, anti-p21 sc, anti-β-actin sc were used. 5% BSA prepared in 0.5% Tris-buffered saline Tween−20 (TBS-T) was used as the blotting agent. Anti-mouse or anti-rabbit antibodies were used as secondary antibodies. Secondary antibodies were diluted 5% BSA, 0.5% TBS-T and incubated at 25οC. Chemiluminescence imaging was used to visualize the membranes.
Production of codon-optimized Human papillomavirus type 52 L1 virus-like particles in Pichia pastoris BG10 expression system
Published in Preparative Biochemistry & Biotechnology, 2023
Kartika Sari Dewi, Sheila Chairunnisa, Sri Swasthikawati, Dian Fitria Agustiyanti, Apon Zainal Mustopa, Wien Kusharyoto, Ratih Asmana Ningrum
In SDS-PAGE analysis, the protein supernatant from the previous step was characterized using 10% acrylamide gel then colored by Coomassie blue R-250 staining. For Western blot analysis, the separated proteins in acrylamide gel were transferred to a nitrocellulose membrane in transfer buffer (25 mM Tris base, 192 mM Glycine, 20% methanol) using Bio-Rad Mini Trans-Blot® at 100 V for 90 min. The membrane was incubated in a blocking buffer containing 5% skim milk in Tris-buffered saline (TBS) pH 7.6 for 1 h at room temperature with gentle shaking. Subsequently, the membrane was washed three times with TBS-T (0.1% Tween-20 in TBS) each for 10 min and incubated with rabbit anti-HPV52 L1 antibody (1:2000 dilution, Creative Diagnostics, New York, US) for 2 h. After the membrane was washed with TBS-T, it was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:3500 dilution, Abnova) for 1 h. At the final stage, the membrane was rewashed in TBS-T and visualized using Tetramethylbenzidine (TMB) substrate for HRP.
Polyclonal antibody-based immunoassay of vitellogenin in Van fish (Alburnus tarichi)
Published in International Journal of Environmental Health Research, 2021
Elif Kaval Oğuz, Kerem Özdemir, Güler Ünal, Ahmet R. Oğuz
Sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using discontinuous gels with 4 and 7.5% polyacrylamide for stacking and resolving gels, respectively. The samples were mixed with equal volumes of the sample buffer. The purified Van fish VTG and plasma from female, male, and E2-injected male were electrophoresed. The gel was stained with Coomassie Brilliant Blue R 250 solution. Molecular weight of VTG was calculated after plotting the logarithm of the molecular weight for the standard proteins (Prestained Protein Ladder, Thermo Scientific). Western blot analysis was performed as previously reported by Shao et al. (2005) with minor modifications. Briefly, Proteins were transferred from gels onto a PVDF membrane by semidry blotting. Nonspecific binding of the membrane was blocked with 5% dry nonfat milk in TBST (Tris-buffered saline, 0.1% Tween 20, pH 7.4) and kept overnight at 4°C. The membrane was then incubated with primary antibody (2 h) at 1:1000 dilution in blocking buffer, followed by incubation with the second antibody solution (peroxidase-conjugated goat anti-rat IgG, 1:1.500 in pH 7.5 Tris-HCl buffer) for 2 h. The antigen-antibody complexes were detected by the peroxidase reaction with 3′,3′-diaminobenzidine and the reaction was terminated after 5 min.