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Two-dimensional and Three-dimensional Dosimetry
Published in W. P. M. Mayles, A. E. Nahum, J.-C. Rosenwald, Handbook of Radiotherapy Physics, 2021
Mark Oldham, Devon Godfrey, Titania Juang, Andrew Thomas
In the early 1990s Maryanski et al. (1994, 1996) developed the first polymer gel dosimeter. It was a polyacrylamide gel (PAG). The initial PAG composition was called the BANG®* gel, as its constituents by weight were bis (3%), acrylamide (3%), nitrogen and gelatin (5%). On irradiation, polymerisation and cross-linking occurs between the acrylic and bis monomers. PAGs are subject to radiation-induced polymerisation of monomers, whereby small monomer molecules join together under the influence of ionising radiation. The resultant long polymer chains are too big to move through the gel lattice, which eliminates image degradation due to diffusion.
Diagnostic applications of immunology
Published in Gabriel Virella, Medical Immunology, 2019
Ajay Grover, Virginia Litwin, Gabriel Virella
Western blotting (Figure 15.3) is a technique that identifies specific proteins in a given sample after their separation using polyacrylamide gel electrophoresis. The polyacrylamide gel is placed adjacent to a membrane, which is typically nitrocellulose or PVDF (polyvinylidene fluoride), and the application of an electrical current induces the proteins to migrate from the gel and immobilize on the membrane. The membrane is subsequently stained with an antibody. The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as discussed previously. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary antihuman antibody linked to an enzyme signal is added. The stained bands indicate the proteins to which the patient's serum is specific. This technique has the advantage over other screening assays of not only detecting antibodies, but also identifying their target antigens, resulting in increased specificity. However, the procedure is of low throughput and requires specialized personnel in certified laboratories. To circumvent these issues, antigen-specific blots that need only be probed with the patient serum and developed, are now commercially available. Using these commercially available kits, the testing can be conducted in general laboratories.
Molecular Biology
Published in John C Watkinson, Raymond W Clarke, Louise Jayne Clark, Adam J Donne, R James A England, Hisham M Mehanna, Gerald William McGarry, Sean Carrie, Basic Sciences Endocrine Surgery Rhinology, 2018
Michael Kuo, Richard M. Irving, Eric K. Parkinson
Negatively charged phosphate groups on the DNA backbone confer a net negative charge on linear DNA. This allows fragments of different sizes to be resolved within a suitable gel matrix by the application of an electric current across the matrix. The DNA will migrate towards the positive electrode with the smaller fragments travelling faster than the larger fragments.10 The size of the fragment can be estimated by the use of a graduated DNA ladder containing fragments of known molecular weight. The choice of the particular matrix depends on the fragment sizes that one is trying to resolve. Polyacrylamide gels can resolve differences of just one base pair between fragments of several hundred base pairs in size by virtue of a small pore size in the gel matrix. These gels can be used for DNA sequencing and resolution of alleles varying in only one dinucleotide repeat. Agarose gels can resolve fragment sizes from around 100 bp to 20 kb. Beyond that size, electrophoretic mobility is no longer proportional to fragment size. Resolution of fragments sizes in excess of 50 kb, such as larger bacterial artificial chromosomes (BAC) or yeast artificial chromosomes (YAC) require the use of pulsed field electrophoresis.
Safflor yellow A protects vascular endothelial cells from ox-LDL-mediated damage
Published in Journal of Receptors and Signal Transduction, 2022
Hu Zhang, Li-Juan Fan, Jun Liu, Jia-Qi Zhu, Ting-Ting Tan, Ming Li, You-Li Zhou
The protein used for loading was obtained from the cells using radioimmunoprecipitation analysis (RIPA) lysis buffer (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The harvested protein supernatant was quantified using BCA protein detection kit (Beyotime, Shanghai, China). Forty micrograms of total protein was loaded into the denatured polyacrylamide gel, passed through the concentrated gel and separating gel in turn, and finally separated according to the molecular weight. In the gel lane, the proteins arranged in order of molecular weight were transferred to the polyvinylidene fluoride (PVDF) membrane. After the nonspecific amino acid residues are sealed with skim milk, the PVDF membrane loaded with the target protein is incubated with the primary antibody. The main antibodies used are as follows: rabbit anti-AMPKα (5832; CST); rabbit anti-p-AMPKα (Thr172) (2535; CST); rabbit anti-GAPDH (A19056; Abclonal). After the incubation, the PVDF membrane was incubated with goat anti-rabbit IgG (H L) secondary antibody (A16096; Thermofisher). The substrate used for the chemical reaction of horseradish peroxidase (HRP) is dropped onto the surface of the PVDF membrane and the luminescent signal is collected. GAPDH was tested in parallel each time to normalize the data of the experimental group and the control group.
Effects of physical exercise combined with captopril or losartan on left ventricular hypertrophy of hypertensive rats
Published in Clinical and Experimental Hypertension, 2021
Quênia Janaína Tomaz de Castro, Carolina Morais Araujo, Patrícia Yoshie Watai, Samara Stéfani de Castro e Silva, Wanderson Geraldo de Lima, Lenice Kappes Becker, Jamille Locatelli, Homero Nogueira Guimarães, Andrea Grabe-Guimarães
Gelatin zymography was used to quantify MMP-2 enzymatic activity in LV tissue as previously described (46). Firstl, LV storage tissues (−80°C) were sprayed with liquid nitrogen (−165°C), then weighed and homogenized in RIPA buffer plus protease inhibitor cocktail (1:1000) at 4°C. After centrifugation (10 min, 10.000 g), the supernatant was used immediately for proteins quantification by using the BCA method (Bio-Rad Protein Assay Kit) as proposed before (47). Thus, the amount of protein was the same for all sample/well. In brief, the tissue extracts were diluted with 10 mM HEPES (pH 7.4) and mixed with a non-reducing loading buffer. Samples were run on 8% polyacrylamide gels copolymerized with 2 mg/ml of gelatin. Gels rinsed in 2.5% Triton X-100 (3 × 20 min) were incubated during 18 h at 37°C in a buffer containing 50 mM Tris, 150 mM NaCl, 5 mM CaCl2 and 0.05% NaN3 (pH 7.5). Gels were stained with 0.05% Coomassie brilliant blue G-250 for 3 h, and destained in a 4:8% methanol:acetic acid solution. After bleaching gels, tagged bands were quantified using Quantity One software (BioRad) using an image capture station (Carestream 4000 MM Pro Image Station, United Kingdom) and the ImageJ software version 1.32 j, by measuring the optical density of each band. All bands were normalized and the results were expressed as band density.
Selective release of circRNAs in platelet-derived extracellular vesicles
Published in Journal of Extracellular Vesicles, 2018
Christian Preußer, Lee-Hsueh Hung, Tim Schneider, Silke Schreiner, Martin Hardt, Anna Moebus, Sentot Santoso, Albrecht Bindereif
For glyoxal Northern blot 5 µg of total RNA from platelets was mixed with glyoxal loading dye (Ambion) and incubated at 50°C for 30 min. RNA was separated by agarose gel electrophoresis (1.2% in 1x MOPS buffer), using the 0.1–2 kb RNA ladder (Thermo Fisher Scientific). The gel was transferred onto a nylon membrane by semidry blotting. RNA was crosslinked by ultraviolet (UV) light and probed with single-stranded RNA probe (DIG RNA Labeling Mix; Roche), which covered the circ-junction. Hybridisation was performed in NorthernMax buffer (Thermo Fisher Scientific), and washes of the blot and probe detection with alkaline phosphatase-conjugated anti-DIG-Fab fragments were done as described in the Roche manual (Roche). RNase H assays were performed as described previously [15]. Subsequent Northern hybridisation analysis on denaturing polyacrylamide gels was done as described above.