Experimental Systems CHAPTER 23 533 for each protein there is a particular pH, called its isoelectricAcidic point(pD), at which that protein has equal numbers of posi- tive and negative charges. Isoelectric focusing makes use of a gel containing substances, called carrier ampholytes, that al range themselves into a continuous pH gradient when sub- jected to an electric field. When a mixture of proteins is ap to such a gel and subjected to electrophoresis, each ein moves until it reaches that point in the gradient the pH of the gel is equal to its isoelectric point. It then stops moving because it has a net charge of zero. Isoelectric focusing is an extremely gentle and effective way of separat ing different proteins(Figure 23-4c) A method known as two-dimensional gel electrophoresis (2D gel electrophoresis) combines the advantages of SDS- PAGE and isoelectric focusing in one of the most sensitive and discriminating ways of analyzing a mixture of proteins In this method, one first subjects the mixture to isoelectric focusing on an IEf tube gel, which separates the molecules on the basis of their isoelectric points without regard to mol- ecular weight. This is the first dimension. In the next step, FIGURE 23-5 Two-dimensional gel electrophoresis of a5s-methionine one places the IEF gel lengthwise across the top of an sDs. labeled total cell proteins from murine thymocytes. These proteins polyacrylamide slab(that is, in place of the sample wells in were first subjected to isoelectric focusing(direction of migration indi- Figure 23-4a)and runs SDS-PAGE Preparatory to this step, SDSPAG arrow) and then the focused proteins were separated by all proteins have been reacted with SDS and therefore mi- SDS-PAGE(direction of migration indicated by blue arrow). The gel grate out of the IEF gel and through the SDS-PAGE slab ac- was exposed to xray film to detect the labeled proteins. [Courtesy af cording to their molecular weights. This is the second dimen- B A Osborne sion. The position of the proteins in the resulting 2D gel car be visualized in a number of ways. In the least sensitive the gel is stained with a protein-binding dye(such as Coomassie blue). If the proteins have been radiolabeled, the more sensi- silver staining is a method odphy can be used. Alternatively, microscope, the theoretical limit of resolution of the electron silver staining is a method of great sensitivity that takes ad- microscope is about 0.002 nm. If it were possible to build an vantage of the capacity of proteins to reduce silver ions to an instrument that could actually approach this limit, the elec- easily visualized deposit of metallic silver. Finally, immuno- tron microscope could readily be used to determine the blotting-blotting of proteins onto a membrane and detec- detailed atomic arrangement of biological molecules, since tion with antibody (see Figure 6-13)-can be used as a way of the constituent atoms are separated by distances of 0. 1 nm to locating the position of specific proteins on 2D gels if an ap- 0.2 nm. In practice, aberrations inherent in the operation of propriate antibody is available. Figure 23-5 shows an autora- the magnetic lenses that are used to image the electron beam diograph of a two-dimensional gel of labeled proteins from limit the resolution to about 0. 1 nm(1A). This practical limit murine thymocytes. can be reached in the examination of certain specimens, par- ticularly metals. Other considerations, however, such as X-Ray Crystallography Provides specimen preparation and contrast, limit the resolution for Structural Information biological materials to about 2 nm(20 A). To determine the arrangement of a molecule's atoms, then, we must turn to A great deal of information about the structure of cells, parts x-rays, a form of electromagnetic radiation that is readily of cells, and even molecules has been obtained by light micro- generated in wavelengths on the order of size of interatomic scopy. The microscope uses a lens to focus radiation to form distances. Even though there are no microscopes with lenses an image after it has passed through a specimen. However, a that can focus x-rays into images, x-ray crystallography can practical limitation of light microscopy is the limit of resolu- reveal molecular structure at an extraordinary level of detail. tion Radiation of a given wavelength cannot resolve struc- X-ray crystallography is based on the analysis of the diffrac tural features less than about 1/2 its wavelength. Since the tion pattern produced by the scattering of an x-ray beam as it shortest wavelength of visible light is around 400 nm, even passes though a crystal. The degree to which a particular atom the very best light microscopes have a theoretical limit of res- scatters x-rays depends upon its size. Atoms such as carbon, olution of no less than 200 nm. oxygen, or nitrogen, scatter x-rays more than do hydrogen Because of the much shorter wavelength(0.004 nm)of atoms, and larger atoms, such as iron, iodide, or mercury give the electron at the voltages normally used in the electron intense scattering X-rays are a form of electromagnetic waves:for each protein there is a particular pH, called its isoelectric point (pI), at which that protein has equal numbers of posi￾tive and negative charges. Isoelectric focusing makes use of a gel containing substances, called carrier ampholytes, that ar￾range themselves into a continuous pH gradient when sub￾jected to an electric field. When a mixture of proteins is ap￾plied to such a gel and subjected to electrophoresis, each protein moves until it reaches that point in the gradient where the pH of the gel is equal to its isoelectric point. It then stops moving because it has a net charge of zero. Isoelectric focusing is an extremely gentle and effective way of separat￾ing different proteins (Figure 23-4c). A method known as two-dimensional gel electrophoresis (2D gel electrophoresis) combines the advantages of SDS￾PAGE and isoelectric focusing in one of the most sensitive and discriminating ways of analyzing a mixture of proteins. In this method, one first subjects the mixture to isoelectric focusing on an IEF tube gel, which separates the molecules on the basis of their isoelectric points without regard to mol￾ecular weight. This is the first dimension. In the next step, one places the IEF gel lengthwise across the top of an SDS￾polyacrylamide slab (that is, in place of the sample wells in Figure 23-4a) and runs SDS-PAGE. Preparatory to this step, all proteins have been reacted with SDS and therefore mi￾grate out of the IEF gel and through the SDS-PAGE slab ac￾cording to their molecular weights. This is the second dimen￾sion. The position of the proteins in the resulting 2D gel can be visualized in a number of ways. In the least sensitive the gel is stained with a protein-binding dye (such as Coomassie blue). If the proteins have been radiolabeled, the more sensi￾tive method of autoradiography can be used. Alternatively, silver staining is a method of great sensitivity that takes ad￾vantage of the capacity of proteins to reduce silver ions to an easily visualized deposit of metallic silver. Finally, immuno￾blotting—blotting of proteins onto a membrane and detec￾tion with antibody (see Figure 6-13)—can be used as a way of locating the position of specific proteins on 2D gels if an ap￾propriate antibody is available. Figure 23-5 shows an autora￾diograph of a two-dimensional gel of labeled proteins from murine thymocytes. X-Ray Crystallography Provides Structural Information A great deal of information about the structure of cells, parts of cells, and even molecules has been obtained by light micro￾scopy. The microscope uses a lens to focus radiation to form an image after it has passed through a specimen. However, a practical limitation of light microscopy is the limit of resolu￾tion. Radiation of a given wavelength cannot resolve struc￾tural features less than about 1/2 its wavelength. Since the shortest wavelength of visible light is around 400 nm, even the very best light microscopes have a theoretical limit of res￾olution of no less than 200 nm. Because of the much shorter wavelength (0.004 nm) of the electron at the voltages normally used in the electron microscope, the theoretical limit of resolution of the electron microscope is about 0.002 nm. If it were possible to build an instrument that could actually approach this limit, the elec￾tron microscope could readily be used to determine the detailed atomic arrangement of biological molecules, since the constituent atoms are separated by distances of 0.1 nm to 0.2 nm. In practice, aberrations inherent in the operation of the magnetic lenses that are used to image the electron beam limit the resolution to about 0.1 nm (1Å). This practical limit can be reached in the examination of certain specimens, par￾ticularly metals. Other considerations, however, such as specimen preparation and contrast, limit the resolution for biological materials to about 2 nm (20 Å). To determine the arrangement of a molecule’s atoms, then, we must turn to x-rays, a form of electromagnetic radiation that is readily generated in wavelengths on the order of size of interatomic distances. Even though there are no microscopes with lenses that can focus x-rays into images, x-ray crystallography can reveal molecular structure at an extraordinary level of detail. X-ray crystallography is based on the analysis of the diffrac￾tion pattern produced by the scattering of an x-ray beam as it passes though a crystal. The degree to which a particular atom scatters x-rays depends upon its size. Atoms such as carbon, oxygen, or nitrogen, scatter x-rays more than do hydrogen atoms, and larger atoms, such as iron, iodide, or mercury give intense scattering. X-rays are a form of electromagnetic waves; Experimental Systems CHAPTER 23 533 Acidic Basic FIGURE 23-5 Two-dimensional gel electrophoresis of 35S-methionine labeled total cell proteins from murine thymocytes. These proteins were first subjected to isoelectric focusing (direction of migration indi￾cated by red arrow) and then the focused proteins were separated by SDS-PAGE (direction of migration indicated by blue arrow). The gel was exposed to x-ray film to detect the labeled proteins. [Courtesy of B. A. Osborne.]