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Characterization of Phyto-Constituents
Published in Rohit Dutt, Anil K. Sharma, Raj K. Keservani, Vandana Garg, Promising Drug Molecules of Natural Origin, 2020
Himangini, Faizana Fayaz, Anjali
In the early 1980s, capillary electrophoresis (CE) was developed as a powerful analytical and separation device. It detects the purity/complexity of a sample and can deal with every kind of charged components of sample from simple inorganic ions to DNA. Thusly, the utilization of fine electrophoretic techniques expanded in the investigation of natural drugs in last past years. The working of CE examination can be performed by electric field worked in tight cylinders which prompts division of numerous mixes. The separation of different charged components caused due to applied voltage in between buffer filled capillaries which generates the production of ions depending on their mass and charge ratio. Frequently used electrophoresis techniques are capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), and capillary isoelectric focusing (CIEF). CE is the most proficient strategy utilized for the division and investigation of modest number of analytes with excellent partition capacity. In the meantime it has comparative specialized qualities as that of liquid chromatography; anyway it is a superior technique for building up the chemical fingerprints of the natural medications.
Haematological disorders
Published in Judy Bothamley, Maureen Boyle, Medical Conditions Affecting Pregnancy and Childbirth, 2020
However, it is recommended that a family origin questionnaire is completed at the booking appointment for each pregnancy. Details are required from both biological parents for ancestry going back for at least 2 generations if possible. The FOQ is used by laboratory staffto conduct screening investigations and to analyse any subsequent DNA tests.30. The blood test for sickle cell disease and other variants is known as electrophoresis.
Autoradiography
Published in Joan Gil, Models of Lung Disease, 2020
At the other end of the spectrum, macroautoradiographs are very useful in combination with the blotting techniques associated with biochemical isolations of proteins, DNA, and RNA. These include single- and double-dimension chromatography, electrophoresis, isoelectric focusing and others. This technology enables the investigator to use a radioactively labeled precursor molecule that can be bound or incorporated into specific molecular species such as DNA, RNA, protein, enzyme, lipids, or carbohydrate in situ. This label can be recovered by various biochemical separations to identify specific molecules or fractions it has been bound to or incorporated. An alternative technique would be to perform separations using the various biochemical techniques and then use radioactively labeled antibodies or DNA or RNA hybridization techniques to identify specific molecules, molecular components, and DNA or RNA sequences or loci.
HBx-induced PLA2R overexpression mediates podocyte pyroptosis through the ROS-NLRP3 signaling pathway
Published in Renal Failure, 2023
Moxuan Feng, Yani Yu, Yueqi Chen, Xiaoqian Yang, Baoshuang Li, Wei Jiang
First, the total cell protein extract was prepared. The protein concentration was determined using a BCA kit for protein concentration determination (Beyotime Biotechnology, Shanghai, China), after which the protein extract was fully denatured with 5× protein loading buffer. Subsequently, electrophoresis was performed using an SDS–PAGE gel preparation kit (Servicebio, Wuhan, China). The proteins on the separating gel were transferred to a PVDF membrane (Millipore, MA, USA). The membrane was then incubated with 5% skim milk and destained at room temperature for 60 min. The membranes were mixed with primary antibodies against PLA2R (ABclonal Technology, Boston, MA, USA, 1:1000, Cat. No.: A10068), NLRP3 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 19771-1-AP), ASC (ABclonal Technology, Boston, MA, USA, 1:1000, Cat. No.: sc-22514-R), caspase-1 (Proteintech, Chicago, IL, USA, 1:5000, Cat. No.: 22915-1-AP), IL-1β (ABclonal Technology, Boston, MA, USA, 1:5000, Cat. No.: A1112), IL-18 (ABclonal Technology, Boston, MA, USA, 1:5000, Cat. No.: A1115), or GAPDH (Proteintech, Chicago, IL, USA, 1:50000, Cat. No: 60004-1-lg) and incubated overnight at 4 °C. After three washes with PBST, the membranes were incubated with fluorescently conjugated goat anti-mouse secondary antibody (ABclonal Technology, Boston, MA, USA, 1:10,000) for 1 h. The target bands were analyzed using Photoshop 2021 and ImageJ software processing systems.
The tumour/normal tissue ratio of Keap1 protein is a predictor for lymphovascular invasion in colorectal cancer: a correlation study between the Nrf2 and KRas pathways
Published in Biomarkers, 2022
Liang-Che Chang, Chung-Wei Fan, Wen-Ko Tseng, Jim-Ray Chen, Chung-Ching Hua
The primary antibodies used were anti-Nrf2 (sc-722; Santa Cruz Biotech, CA, USA; 1:900 dilution), anti-Keap1 (sc-33569; Santa Cruz; 1:600), anti-Bach1 (NBP1-88722; Novus Biologicals, Littleton, CO, USA; 1:250), anti-p62 (ab-109012; Abcam, Cambridge, MA, USA; 1:1000), anti-HO1 (ab-13243; Abcam; 1:600), anti-KRas (SC-30; Santa Cruz; 1:1000), anti-Erk1/2 (SC-514302; Santa Cruz; 1:1000), anti-PI3K (SC-1637; Santa Cruz; 1:600), anti-Raf1 (SC-7267; Santa Cruz; 1:1000) and anti-β-actin (sc-47778; Santa Cruz; 1:600). The secondary antibodies were obtained from Santa Cruz (sc-2005) and Abcam (ab-6721). Gel electrophoresis was done as previously described (Chang et al.2018). In brief, the proteins were separated on a 12% Tris-glycine PAGE gel and then transferred to a PVDF membrane which was subsequently incubated first with primary antibodies at 4 °C overnight and then with secondary ones for 1 hour at room temperature. The protein levels were detected by chemiluminescence and visualised. The level of each protein was normalised to that of β-actin. An example of protein electrophoresis is illustrated in Figure 2.
Recent developments in Phos-tag electrophoresis for the analysis of phosphoproteins in proteomics
Published in Expert Review of Proteomics, 2022
The term ‘proteome’ was coined by Marc R. Wilkins in 1995 [27]. Around that time, proteome analysis was mainly based on 2-DE and MS as described above. Various analytical techniques necessary for performing proteome analysis via electrophoresis have been developed and improved since the 1990s [28–31]. Using these techniques, many exciting proteins, including disease-related and stress-induced proteins, have been discovered [32–39]. However, the greater the number of proteins separated using electrophoresis, the greater the time and effort required to identify the proteins. Additionally, there were limitations with regard to the protein species that can be separated via electrophoresis in general laboratories. Thus, development of a technique capable of performing more efficient analysis has been a necessity.