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Approaches for Identification and Validation of Antimicrobial Compounds of Plant Origin: A Long Way from the Field to the Market
Published in Mahendra Rai, Chistiane M. Feitosa, Eco-Friendly Biobased Products Used in Microbial Diseases, 2022
Lívia Maria Batista Vilela, Carlos André dos Santos-Silva, Ricardo Salas Roldan-Filho, Pollyanna Michelle da Silva, Marx de Oliveira Lima, José Rafael da Silva Araújo, Wilson Dias de Oliveira, Suyane de Deus e Melo, Madson Allan de Luna Aragão, Thiago Henrique Napoleão, Patrícia Maria Guedes Paiva, Ana Christina Brasileiro-Vidal, Ana Maria Benko-Iseppon
An excellent criterion for selecting the solution for the extraction of globular proteins is the relationship between protein quantity and its biological activity. Thus, the protein purifier must avoid producing an inactive crude extract even if it contains a high protein concentration or an active extract, but with a minimum amount of the targeted protein. Protein detection can be done through absorbance at 280 nm or by colorimetric methods as Bradford, Lowry, Biuret, and bicinchoninic acid (Zheng et al. 2017; Silva et al. 2019; Licini et al. 2020).
Biocompatibility and Biomaterials
Published in Sirshendu De, Anirban Roy, Hemodialysis Membranes, 2017
Protein content is estimated via the bicinchoninic acid (BCA) assay.12 When placed in alkaline solution containing Cu2+ ions proteins, it forms a colored complex between the peptide bonds and copper atoms. This direct technique was, however, inefficient to detect low concentrations and hence the Lowry assay was developed, where the Folin–Ciocalteu reagent was used to enhance color. BCA is a substitute for the Folin–Ciocalteu reagent forming a 2:1 complex with protein resulting in a stable and highly colored complex detectable at 562 nm.12
Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
For protein isolation, samples stored at −80°C are first pulverised in liquid nitrogen (LN2) prior to homogenisation on ice, using handheld or machine homogenisers (described above) in relevant lysis buffer (depending on the analyses to be performed e.g. Western blotting and proteomics as described later in this chapter), typically contain protease and phosphatase inhibitors, that prevent proteins from degrading and halt any continued phosphorylation. Preparation on ice is also important (in combination with phosphatase inhibitors), particularly if phospho-protein analyses are to be performed. Following homogenisation, samples are centrifuged at, e.g., 1,000 g at 4 °C for 5 minutes. Soluble proteins are contained within the supernatant and non-soluble (e.g. myofibrillar proteins) in the ensuing pellet. The pellet can be resuspended in relevant assay buffer and protein concentrations determined by specific protein assays such as the bicinchoninic acid (BCA) or Bradford protein assay, prior to aliquoting for long-term storage or for further analyses. As an example, the BCA protein assay detects Cu+1, and is created when Cu+2 is reduced by protein in an alkaline environment (14). When the reagent supplied in the BCA assay is mixed with the protein sample, two molecules of BCA join with one cuprous (Cu+1) molecule and the reaction becomes purple-coloured. This purple colour is due to the macromolecular structure of protein, the number of protein bonds and the presence of the four amino acids: Cysteine, Cystine, Tryptophan and Tyrosine (15). The darker the colour, the more protein present. The absorbance of this complex is then measured using a spectrometer between 540 and 590 nM which is linear with increasing protein concentrations.
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.
ROBO2 hampers malignant biological behavior and predicts a better prognosis in pancreatic adenocarcinoma
Published in Scandinavian Journal of Gastroenterology, 2021
Cheng Ding, Yatong Li, Shunda Wang, Cheng Xing, Lixin Chen, Hanyu Zhang, Yizhi Wang, Menghua Dai
Cultured cells were lysed in RIPA buffer supplemented with protein phosphatase inhibitor (P1260, Applygen Technologies, Beijing) and proteinase inhibitor cocktail (P1265, Applygen Technologies, Beijing) on ice for 0.5h and centrifuged 15 min (10000 rpm). We use bicinchoninic acid kit to evaluate protein concentration. The lysate containing approximately 20.0 µg of protein was loaded onto 8% SDS-PAGE gel and then run for 2.5 h at 80 V. Proteins were transferred to Immobilon-PVDF membranes (EMD Millipore, Burlington, MA, USA) at 400 mA for 1.5 h. The membranes were sealed with 5% skim milk, then we incubated the membrane with the following primary antibodies: anti-ROBO2 antibody at 1:1000 dilution (ab75014; Abcam) and anti-GAPDH antibody at 1:2000 dilution (60004-1-Ig; Proteintech, Rosemont, IL, USA). In addition, the following antibodies were used:anti-ECM1 antibody (11521-1-AP; Preoteintech), anti-N-cadherin antibody (13116; Cell Signaling Technology, Danvers, MA, USA), anti-E-cadherin antibody (14472; Cell Signaling Technology), and anti-vimentin antibody (5471; Cell Signaling Technology).