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Molecular Analysis in Mechanobiology
Published in Jiro Nagatomi, Eno Essien Ebong, Mechanobiology Handbook, 2018
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting is analogous to Northern blotting for mRNA analysis, providing a separation on the basis of molecular weight and identification/semi-quantitative measurements of specific proteins using antibodies. Samples may be prepared from biological fluids, cell culture supernatants, or cell lysates. The technique is particularly useful for studying intracellular and transmembrane proteins with limited solubility in aqueous solutions. Samples are mixed with a concentrated loading buffer containing SDS, heated (usually 10 min at 60°C), and loaded into wells formed in covalently cross-linked polyacrylamide gels [69]. Heating in the presence of SDS denatures the proteins, reducing secondary structure so that their migration in the gel will be primarily a function of molecular weight, while SDS binding provides a net negative charge to all proteins. It is also common to include beta-mercaptoethanol in the loading buffer to reduce disulfide bonds (reducing conditions). A “ladder” composed of proteins of known molecular weight is loaded in one well as a basis for molecular weight determination. Polyacrylamide gels are usually prepared with a top “stacking” region containing the sample wells and a bottom “resolving” region. Proteins migrate rapidly through the stacking region and are concentrated at the interface before entering the resolving region. The acrylamide concentration in the resolving gel can be varied to improve resolution within certain molecular weight ranges (8% for the 100–200 kDa range; 12% gels for the 10–40 kDa range).
Proteins and proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Native polyacrylamide gel electrophoresis (PAGE) is an electrophoretic separation method typically used in proteomics and metallomics. Native PAGE separations are run in non-denaturing conditions. Detergents are used only to the extent that they are necessary to lyse lipid membranes in the cell. Complexes remain—for the most part—associated and folded, as they would be in the cell. One downside, however, is that complexes may not separate cleanly or predictably, since they cannot move through the poly acrylamide gel as quickly as individual, denatured proteins. Take care not to confuse native PAGE with SDS-PAGE (sodium dodecyl sulfate PAGE). SDS-PAGE uses a detergent, sodium dodecyl sulfate, to denature the proteins and provide a negative charge that is proportional to the protein mass, allowing elec-trophoretic separation. There are three popular methods of native PAGE: blue native (BN-PAGE), clear native (CN-PAGE), and quantitative preparative native continuous (QPNC-PAGE) (Figure 3.13).
Principles and Techniques for Deoxyribonucleic Acid (DNA) Manipulation
Published in Hajiya Mairo Inuwa, Ifeoma Maureen Ezeonu, Charles Oluwaseun Adetunji, Emmanuel Olufemi Ekundayo, Abubakar Gidado, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi, Medical Biotechnology, Biopharmaceutics, Forensic Science and Bioinformatics, 2022
Nwadiuto (Diuto) Esiobu, Ifeoma M. Ezeonu, Francisca Nwaokorie
PAGE (polyacrylamide gel electrophoresis) is a type of gel technique often used to separate components of a protein mixture based on their size. Acrylamide is the gel matrix, instead of agarose. It is soluble in water, and readily polymerizes in solution, resulting in the formation of polyacrylamide. To induce the polymerization of acrylamide and bis-acrylamide monomers, ammonium persulfate (APS), which spontaneously decomposes to form free radicals, is added to the gel. TEMED, a free radical stabilizer, is also included to promote polymerization. As in agarose gels, increased concentrations of acrylamide results in decreased pore size after polymerization. The experimenter determines the concentration of the polyacrylamide gel based on the size of the molecules being studied. Like all gel electrophoresis techniques, PAGE is based on the principle that a charged molecule will migrate in an electric field towards an electrode with opposite charge. However, to accurately determine the molecular weight of biological molecules, the mobility of a substance in the gel cannot depend on both charge and size since the varying charges complicate the rate of migration through the matrix! So, during PAGE, the biological samples are pre-treated so that they acquire uniform charge, allowing the electrophoretic mobility to be driven primarily by size. Different protein molecules with differing shapes and sizes are denatured with the aid of an anionic detergent – SDS whose interaction with proteins results in the loss of their secondary, tertiary or quaternary structures. The proteins mixed with SDS are uniformly negatively charged. Loading such samples onto a gel in a vertical electric field (created by the power supply) allows migration towards the anode at a rate determined by a molecular sieving that is based on size (Figure 1.3c). Upon completion of the run, gels are visualized after staining and destaining techniques, with dyes such as Coomasie brilliant blue. The size of a protein can be calculated by comparing its migration distance with that of a known molecular weight ladder.
Expression, purification, and characterization of N-terminal His-tagged proteins with mutations in zinc finger 3 of zinc finger protein ZNF191(243–368)
Published in Preparative Biochemistry and Biotechnology, 2018
Dongxin Zhao, Zhongxian Huang, Jie Liu, Li Ma, Juan He
The optimum conditions for the expression of the recombinant proteins in the E. coli M15 host bacterium were determined. High levels of protein expression were induced under the optimum conditions. Bacteria were grown to OD600=0.6 at 37 °C, and induced by 0.1 mM IPTG for 6 hr. The expression and purification conditions of the mutant proteins were similar to those of His6-ZNF191(243–368).[6,7] The cells harvested from 500 mL of the LB medium grown and induced under the optimum conditions were suspended in 10 volumes of cell lysis buffer (50 mmol·L−1 NaH2PO4, pH 8.0, 300 mmol·L−1 NaCl, 5 mmol·L−1 imidazole). The cells were lysed by lysozyme for about 1 hr at 4 °C, treated with 5 U·mL−1 DNase, and stirred for 30 min to degenerate nucleic acids at 4 °C. Soluble and insoluble cell fractions were separated by centrifugation at 15,000 rpm for 30 min. The supernatants were mixed with Ni-NTA resin to purify fusion proteins in accordance with the manufacturer’s manual. The His-tagged proteins were eluted in an elution containing 50 mmol·L−1 NaH2PO4, 300 mmol·L−1 NaCl, and 250 mmol·L−1 imidazole at pH 8.0. The eluted solution was concentrated using Amicon YM-5, and passed through a Sephadex G-75 column to remove the impurities and through a Sephadex G-25 column to remove salts. The collected protein solution was then lyophilized. The purified proteins were mixed with 2 × sodium dodecyl sulfate loading buffer and boiled for 10 min to prepare the samples. The samples were then detected by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Coomassie Brilliant Blue R-250 stainning. Target proteins were detected by comparison with protein standard markers.