Chapter 2 Electrophoresis Techniques Summarize The introduction of

Chapter 2 Electrophoresis Techniques

Summarize The introduction of electrophoresis techniques The classification of electrophoresis techniques The principles and operations of electrophoresis techniques The application of electrophoresis techniques (Discussion in the class) 05 March 2021 Genetic Engineering 2

1. 1 The introduction of electrophoresis techniques Electrophoresis is a technique used to separate and sometimes purify macromolecules大分子 - especially proteins and nucleic acids - that differ in size, charge or conformation构象. In contrast to proteins, which can have either a positive or negative charge, nucleic acids have a consistent 一定的 negative charge imparted给予 by their phosphate 磷酸的backbone, and migrate迁移 toward the anode阳极. 05 March 2021 Genetic Engineering 4

1. 1 The introduction of electrophoresis techniques Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the p. H at a relatively constant value. 蛋白质或核酸能够在介质当中进行移动. 最常见的是利用模具 制备具有上样孔的凝胶板，然后将凝胶进入到含有电解质或 能够维持PH值处于相对稳定的电泳缓冲液当中。 05 March 2021 Genetic Engineering 5

1. 2 The classification of electrophoresis techniques According to the difference of the medium Cellulose acetate membrane electrophoresis Agarose gel electrophoresis SDS Polyacrylamide gel electrophoresis Capillary electrophoresis According to the difference of analyzing subjects Nucleic acid electrophoresis Protein or peptides electrophoresis 05 March 2021 Genetic Engineering 6

1. 2. 1 Cellulose acetate membrane electrophoresis 醋酸纤维素膜 The principles of cellulose acetate membrane electrophoresis +H 3 N-P-COOH Positive ion 阳离子 -H+ +H 3 N-P-COO Zwitterion 两性离子 - -H+ +H+ H 2 N-P-COONegative ion 阴离子 The protein which can have either a positive or negative charge in the solution at different p. H. 05 March 2021 Genetic Engineering 8

1. 2. 1 Cellulose acetate membrane electrophoresis 醋酸纤维素膜 The medium Cellulose acetate Composed 由. . 组成of cellulose di 二（词头）and tri三（词头）acetate, it exhibit low static charge 静电荷, high strength and good resistance to heat. 05 March 2021 Magnification of Cellulose Acetate filter Genetic Engineering 9

1. 2. 1 Cellulose acetate membrane electrophoresis 醋酸纤维素膜 The electrophoresis apparatus of cellulose acetate membrane 05 March 2021 Genetic Engineering 10

1. 2. 1 Cellulose acetate membrane electrophoresis 醋酸纤维素膜 The electrophoresis apparatus of cellulose acetate membrane 05 March 2021 Genetic Engineering 15

1. 2. 1 Cellulose acetate membrane electrophoresis 醋酸纤维素膜 05 March 2021 Genetic Engineering 16

1. 2. 2 Agarose gel electrophoresis - The principles of agarose gel electrophoresis - + - - The nucleic acid that differ in size. 05 March 2021 Magnification of Agarose gel Like a cribble 筛子 Genetic Engineering Separated by size 17

1. 2. 2 Agarose gel electrophoresis Circular forms of DNA migrate in agarose differently from linear DNAs of the same mass. The plasmids that differ in conformation 05 March 2021 Genetic Engineering uncut 18

1. 2. 2 Agarose gel electrophoresis 05 March 2021 Genetic Engineering 19

1. 2. 2 Agarose gel electrophoresis Let us watch a video about Agarose gel electrophoresis. There are several goals for us to achieve! Understand the principles of Agarose gel electrophoresis. Learn the procedures of Agarose gel electrophoresis. What should be pay more attention when you running a agarose gel? 05 March 2021 Genetic Engineering 20

1. 2. 2 Agarose gel electrophoresis The procedures of agarose gel electrophoresis 05 March 2021 Genetic Engineering 21

The procedures of agarose gel electrophoresis To pour a gel, agarose powder is mixed with electrophoresis buffer to the desired concentration, then heated in a microwave oven until completely melted. Most commonly, ethidium bromide is added to the gel (final concentration 0. 5 ug/ml) at this point to facilitate visualization of DNA after electrophoresis. After cooling the solution to about 60 C, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature or, if you are in a big hurry, in a refrigerator. After the gel has solidified, the comb is removed, using care not to rip the bottom of the wells. The gel, still in its plastic tray, is inserted horizontally into the electrophoresis chamber and just covered with buffer. Samples containing DNA mixed with loading buffer are then pipeted into the sample wells, the lid and power leads are placed on the apparatus, and a current is applied. You can confirm that current is flowing by observing bubbles coming off the electrodes. DNA will migrate towards 05 March 2021 electrode, which Genetic Engineering 22 the positive is usually colored red.

1. 2. 3 SDS-PAGE electrophoresis The principles of SDS-PAGE electrophoresis 05 March 2021 Genetic Engineering 23

1. 2. 3 SDS-PAGE electrophoresis The principles of SDS-PAGE Acrylamide: 丙烯酰胺 Bis: 甲X丙烯酰胺 Ammonium persulfate : 过硫酸铵 TEMED: N, N, N'-四甲基乙二胺 05 March 2021 Genetic Engineering 24

The principles of SDS-PAGE electrophoresis Magnification of PAGE gel 05 March 2021 Genetic Engineering 25

The principles of SDS-PAGE electrophoresis 05 March 2021 Genetic Engineering 26

The principles of SDS-PAGE electrophoresis SDS The protein that differ in size. 05 March 2021 Magnification of PAGE gel Genetic Engineering Separated by size 27

The principles of SDS-PAGE electrophoresis The difference between nondenature gel and SDS-PAGE denature gel 05 March 2021 Genetic Engineering 28

1. 2. 2 SDS-PAGE electrophoresis The procedures of SDS-PAGE electrophoresis Making the SDS-PAGE gel. Running the SDS-PAGE gel. Staining the SDS-PAGE gel. There are three videos about the procedures of SDS-PAGE. In this part we should learn how to make , run and stain the SDS-PAGE gel. 05 March 2021 Genetic Engineering 29

1. 2. 5 other types of electrophoresis Isoelectric focusing，IEF 05 March 2021 Genetic Engineering 31

1. 2. 4 other types of electrophoresis two-dimensional electrophoresis 05 March 2021 Genetic Engineering 32

1. 3 The application of electrophoresis techniques Discussion in the class The electrophoresis techniques are used to separate and analyze the samples which have charge, especially protein and nucleic acids. According to the different purposes , we should choose different electrophoresis techniques. We have learned several kinds of electrophoresis techniques already. Would you please categorize them according to the different purposes? What is your opinion about the application of 05 March 2021 Genetic Engineering 33 electrophoresis techniques in research and our life?