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Preclinical Characterization of Engineered Nanoparticles Intended for Cancer Therapeutics
Published in Mansoor M. Amiji, Nanotechnology for Cancer Therapy, 2006
Anil K. Patri, Marina A. Dobrovolskaia, Stephan T. Stern, Scott E. McNeil
The average number of targeting agents per nanoparticle has to be optimized for both solubility and binding affinity. Affinity chromatography or SEC can be employed with some nanoparticles to separate nanoparticles with targeting agents from those without targeting agents. In nanoparticles containing antibodies19,46 or proteins, quantification can be achieved using an enzyme-linked immunosorbent assay (ELISA) or bicinchoninic acid assay (BCA) if the inherent property of the nanoparticle itself does not interfere with the assay. In the case of dendrimers, NMR has been successfully applied to analyze the average number of targeting agents by comparing the integration values of the signals associated with the targeting agents to those belonging to the dendrimer. This is still an averaged technique that cannot distinguish the distribution of targeting agent density on a population of nanoparticles.
Stigmasterol alleviates allergic airway inflammation and airway hyperresponsiveness in asthma mice through inhibiting substance-P receptor
Published in Pharmaceutical Biology, 2023
Jimei Zhang, Chonghong Zhang, Li Miao, Zimin Meng, Ning Gu, Guifang Song
Total proteins were extracted from BEAS-2B cells and mouse lung homogenates using TRIzol reagent. Bicinchoninic acid assay was performed to quantify protein concentration. Protein sample (30 μg) was separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then shifted to PVDF membranes. After blocking by 5% non-fat milk for 1 h. The blots were incubated with primary antibody neurokinin-1 receptor polyclonal antibody (1:800, 17942-1-AP, Proteintech), GAPDH (1:5000, 10494-1-AP, Proteintech) at 4 °C overnight. Then, PVDF membranes were incubated with secondary antibodies (1:2000, ab6721, Abcam). After washing 3 times with TBST solution, a chemiluminescence (ECL) reagent was used to make proteins visible. The grey value of each band was recorded and analyzed by Image J 1.49p software.
LncRNA AWPPH is downregulated in osteoporosis and regulates type I collagen α1 and α2 ratio
Published in Archives of Physiology and Biochemistry, 2022
Guang Qian, Yueming Yu, Youhai Dong, Yang Hong, Minghai Wang
Total proteins were extracted from cells using an ice-cold RIPA buffer. The bicinchoninic acid assay was performed to determine protein concentrations. An equal amount of protein samples (20 μg) were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories), and blocked in 5% non-fat milk for 1 h, followed by incubation with primary antibodies against RUNX2 (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C for overnight. After three 10-min washes in phosphate-buffered saline, the membranes were incubated with a secondary antibody at room temperature for 1 h. The protein bands were visualised by enhanced chemiluminescence detection and analysed by Image Pro Plus software. The relative protein expression was normalised to GAPDH.
BDNF-overexpressing human umbilical cord mesenchymal stem cell-derived motor neurons improve motor function and prolong survival in amyotrophic lateral sclerosis mice
Published in Neurological Research, 2021
Jie Wang, Weiwei Hu, Zehua Feng, Meijiang Feng
After 30 min of homogenization in RIPA buffer containing Phenylmethylsulfonyl fluoride and centrifugation for 5 min at 10,000 rpm at 4°C, cell or tissue lysates were collected and stored at −80°C. The bicinchoninic acid assay (PIERCE, USA) was used to determine protein concentration. ChAT, HB9, BDNF and hSOD1 expression in cells or lumbar enlargement tissues was detected by western blotting. Briefly, proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, USA), then blocked with 5% skim milk for at least 1 h in Tris buffered saline with tween 20 at room temperature, and were subsequently incubated overnight with anti-ChAT, anti-hSOD1 (Proteintech, USA), anti-BDNF (Abcam, UK), anti-HB9 (Bioss, China), and anti-GAPDH (Yifeixue, China) primary antibodies (1:1000) at 4°C. GAPDH was used as the internal control. After several washes and 90 min incubation at room temperature with HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibody (1:5000, Best, China), electrochemiluminescence solution (Beyotime, China) and an imaging system (Clinx, China) were used to detect the chemiluminescent signals of bound antibodies, the density of which was analyzed by Image J (National Institutes of Health, USA).