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Proteins and proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Two-dimensional gel electrophoresis or 2D electrophoresis is a form of gel electrophoresis commonly used to separate a large number of proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2D electrophoresis begins with ID electrophoresis but then separates the molecules by a second property in the direction 90° from the first. In ID electrophoresis, proteins are separated in one dimension, so that all the proteins lie along a lane, but separated from each other by an isoelectric point. The result is that the molecules are spread out across a 2D gel. Because it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively separated by 2D electrophoresis than in ID electrophoresis. Separating proteins by isoelectric point is called isoelectric focusing. Thereby, a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled toward the more negative end of the gel and if they are negatively charged, they will be pulled to the more positive end of the gel. The proteins applied in the first dimension will move along the gel and will accumulate at their isoelectric point. That is, the point at which the overall charge of the protein is 0 (a neutral charge). The result of this is a gel with proteins spread out on its surface. These proteins can then be detected by a variety of means, but the most commonly used are silver and coomassie staining. In this case, a silver colloid is applied to the gel. The silver binds to cysteine groups within the protein. The silver is darkened by exposure to ultraviolet light. The darkness of the silver can be related to the amount of silver and, therefore, the amount of protein at a given location on the gel. This measurement can only give approximate amounts but is adequate for most purposes (Figure 3.17).
Proteins and Proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
Two-dimensional gel electrophoresis or 2-D electrophoresis is a form of gel electrophoresis commonly used to separate many proteins. Mixtures of proteins are separated by two properties in two dimensions on 2-D gels. 2-D electrophoresis begins with 1-D electrophoresis but then separates the molecules by a second property in the direction 90° from the first. In the 1-D electrophoresis, proteins are separated in one dimension, so that all the proteins will lie along a lane but separated from each other by an isoelectric point. The result is that the molecules are spread out across the 2-D gel. Because it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively separated by 2-D electrophoresis than by 1-D electrophoresis. To separate the proteins by isoelectric point is called isoelectric focusing (IEF). Thereby, a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled towards the more negative end of the gel and if they are negatively charged, they will be pulled to the more positive end of the gel. The proteins applied in the first dimension will move along the gel and will accumulate at their isoelectric point. That is, the point at which the overall charge of the protein is 0 (a neutral charge). The result of this is a gel with proteins spread out on its surface. These proteins can then be detected by a variety of means, but the most commonly used are silver and Coomassie staining. In this case, a silver colloid is applied to the gel. The silver binds to cysteine groups within the protein. The silver is darkened by exposure to UV light. The darkness of the silver can be related to the amount of silver and therefore the amount of protein at a given location on the gel. This measurement can only give approximate amounts but is adequate for most purposes (Figure 3.17).
Downstream Processing
Published in Debabrata Das, Soumya Pandit, Industrial Biotechnology, 2021
where q is the charge on the particle, E is electric field intensity, DP is particle diameter, m is the viscosity of the fluid, and Vt is the terminal velocity of the particle. Depending on the pH of the medium, electrostatic charges on protein molecules will be different. When pH > pI, the protein will be negatively charged; and when pH < pI, the charge on the protein will be positive. The net charge on the protein will determine the velocity of the protein. When a protein molecule is placed in a pH gradient, the electrophoretic velocity becomes zero when pH = pI, since the net charge on the protein is zero at pH = pI. Precipitation of proteins in a pH gradient at their isoelectric point is known as isoelectric focusing. Proteins will be separated from each other in an electric field. Certain gels, such as agar or polyacrylamide, are used for protein separations by gel electrophoresis. Gel electrophoresis is an important analytical separation technique. Scale-up is problematic due to thermal convection resulting from electrical heating. One analytical and micropreparative version of electrophoresis that has an excellent resolution is called two-dimensional protein electrophoresis (2DE). The 2DE procedure is actually the series combination of two electrophoretic separations which resolve protein mixtures based on two independent characteristics – charge and size (Kalyanpur, 2002). Proteins are first separated in a polyacrylamide gel matrix using isoelectric focusing, an equilibrium separation technique that resolves proteins based on their respective isoelectric points. Proteins are subsequently coated with an anionic surfactant and separated by size in another polyacrylamide gel. Finally, proteins are detected using a chemical stain, by autoradiography or by other methods. The result of this two-dimensional separation is a high-resolution fingerprint of protein expression that is characteristic of a particular biological system. This technique has at least two orders of magnitude better resolution than any other analytical tool for protein analysis. Separated protein spots can be cut out of the polyacrylamide gels and subjected to further microchemical analysis to determine the amino acid sequence (Zydney, 2016).
Determination of the isoelectric point of shortened glucagon-like peptide-1 by capillary isoelectric focusing with whole column imaging
Published in Instrumentation Science & Technology, 2018
Junjian Fang, Jiexin Liu, Haijing Li, Jianhua Cheng, Shengming Wu, Fangting Dong
Figure 1 shows a schematic diagram of capillary isoelectric focusing-whole column imaging detection. The inlet of the capillary column is connected to autosampler and the outlet of the capillary column is connected to a waste vial. When high voltage direct current is passed into the cathode and anode electrolyte, a stable pH gradient is established by carrier ampholytes.[11] The sample moves toward the anode or cathode in the 50 mm capillary column based on its charge. When the protein reaches its isoelectric point, which is equal to the pH value, the protein movement stops and the isoelectric focusing is completed. During the focusing process, a complementary metal-oxide-semiconductor camera is used to record the entire dynamic imaging in this capillary column and ultraviolet absorption is used as the detection signal.