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Protein Engineering and Bionanotechnology
Published in Anil Kumar Anal, Bionanotechnology, 2018
The classical method for protein identification involves microsequencing by Edman chemistry. Edman sequencing technique was introduced in 1949 by Edman that identifies N-terminal sequence of amino acid. This method is utilized for the identification of protein separated by SDS-PAGE. The general procedure for mixed-peptide sequencing involves PAGE for the separation of protein mixture followed by transfer of the target protein to an inert membrane by electroblotting. During electroblotting, proteins are visualized on the membrane surface, and excised and fragmented into peptides. The membrane is placed into an automated Edman sequencer where 6–12 automated Edman cycles are carried out. The resulting sequence data are fed into the FASTF (protein database) or TFASTF (DNA database) algorithms, which sort and match the data against stored database and identify the target protein (Graves and Haystead 2002).
History of Aptamer Development
Published in Rakesh N. Veedu, Aptamers, 2017
Nasa Savory, Koichi Abe, Taiki Saito, Kazunori Ikebukuro
While cell-SELEX can target only membrane-associated proteins, aptamers against any unpurified proteins in a crude sample or tissue samples can be obtained by SELEX combined with polyacrylamide gel electrophoresis (PAGE) and electroblotting. Aptamer selection against protein targets separated by native PAGE, sodium dodecyl sulfate (SDS)-PAGE, or two-dimensional electrophoresis from crude samples followed by electroblotting onto a nitrocellulose or polyvinylidene difluoride (PVDF) membrane has been demonstrated; these methods offer potential as powerful tools for novel biomarker discovery [26–28]. Oligonucleotides binding to target proteins on a membrane can be extracted by excision of the membrane region, immobilizing the target protein. By comparing PAGE patterns between samples from normal and abnormal cells or tissues, we can simultaneously identify biomarker candidates and aptamers against the candidate.
The protective mechanisms underlying Ginsenoside Rg1 effects on rat sciatic nerve injury
Published in Journal of Toxicology and Environmental Health, Part A, 2019
Dong-Sheng Huo, Jian-Fang Sun, Zhi-Ping Cai, Xu-Sheng Yan, He Wang, Jian-Xin Jia, Zhan-Jun Yang
Western blot was conducted as previously described by Huo et al. (2016). Protein concentration was measured using a Bio-Rad protein assay kit (Bio-Rad, USA), and sodium dodecy 1 sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of samples was performed with gels comprised of 8% acrylamide and 4% bisacrylamide. Proteins were transferred from gels to polyvinylidene fluoride (PVDF) membranes by electroblotting. The membranes were reacted with a rabbit polyclonal antibody against caspase-3 (1:1000; Abcam, UK) and XIAP (1:1000; Abcam, UK) at 4◦C for 12 hr, respectively, and subsequently incubated with secondary goat anti-rabbit antibody at 37◦C for 1 h. The intensities of immunoreactive bands were quantified by Quantity One software (Bio-Rad, USA). Results are presented as means ± SD in triplicate.
Involvement of apoptosis in the protective effects of Dracocephalum moldavaica in cerebral ischemia reperfusion rat model
Published in Journal of Toxicology and Environmental Health, Part A, 2019
Peng Wu, Xu-Sheng Yan, Li-Li Zhou, Xin-Lang Liu, Dong-Sheng Huo, Wei Song, Xin Fang, He Wang, Zhan-Jun Yang, Jian-Xin Jia
Whole brain tissue sections were also used for immunohistochemical and Western blot analysis analyses as described by Ishii et al. (2012) and Huo et al. (2016). Briefly, the sections obtained were dewaxed and hydrated with 3% hydrogen peroxide, and then treated with antibodies (1:400) for 24 h. Protein concentrations were measured using a Bio-Rad protein assay kit (Bio-Rad, USA), and sodium dodecy1 sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of samples was performed with gels comprised of 8% acrylamide and 4% bisacrylamide. Proteins were transferred from gels to polyvinylidene fluoride (PVDF) membranes by electroblotting. The membranes were reacted with rabbit polyclonal antibodies for p53 (1:1000), bax (1:1000) and bcl-2 (1:1000) at 4°C for 12 h, respectively, then incubated with goat anti-rat secondary antibody at 37°C for 2 h. The intensities of the immunoreactive bands were quantified by Quantity One software (Bio-Rad, USA). On the second day the chemical staining was performed. The positively stained cells were visualized by a microscope at ×100 magnification. Results were presented as mean ± SD in triplicate.
Mechanisms Associated with Protective Effects of Ginkgo Biloba Leaf Extracton in Rat Cerebral Ischemia Reperfusion Injury
Published in Journal of Toxicology and Environmental Health, Part A, 2019
Wei Song, Jun Zhao, Xu-Sheng Yan, Xin Fang, Dong-Sheng Huo, He Wang, Jian-Xin Jia, Zhan-Jun Yang
For low, intermediate or high GbE doses and MCAO alone, the brain tissue around the infarct core area was separated. Similar brain area tissues were taken from control and sham group. The tissue was ground at low temperature with cell lysis solution, protease inhibitor, and ultrasonic dispersion. The homogenate was centrifuged for 30 min at 4°C at 12,000 g, and the supernatant obtained after 24 h. Protein concentrations were measured using a Bio-Rad protein assay kit (Bio-Rad, USA), and separated on 10% SDS-polyacrylamide gels, and transferred to polyvinylidenedifluoride (PVDF) membranes by electroblotting as described previously (Jia et al. 2016, 2018). The strips were cut according to molecular weight. The membranes were reacted with rabbit polyclonal antibodies for TNF-α and IL-6 12 h then incubated overnight with goat anti-rat secondary antibody at 37°C for 2 h. The intensities of the immunoreactive bands were quantified by Quantity One software (Bio-Rad, USA). On the second day, the chemical staining was performed. The positively stained cells were visualized by a microscope at ×100 magnification. Results were presented as mean ± SD in triplicate.