H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 38
Текст из файла (страница 38)
If the solution is allowed toflow across the surface, then molecules that interact frequently with the surface will spend more time bound to thesurface and thus move more slowly than molecules that interact infrequently with the surface. In this technique, calledliquid chromatography, the sample is placed on top of atightly packed column of spherical beads held within a glasscylinder. The nature of these beads determines whether theseparation of proteins depends on differences in mass,charge, or binding affinity.Gel Filtration Chromatography Proteins that differ in masscan be separated on a column composed of porous beadsmade from polyacrylamide, dextran (a bacterial polysaccharide), or agarose (a seaweed derivative), a technique called gelfiltration chromatography.
Although proteins flow around thespherical beads in gel filtration chromatography, they spendsome time within the large depressions that cover a bead’s surface. Because smaller proteins can penetrate into these depres-4.25.9pI7.4dimensional gel of a protein extract from cultured cells, eachspot represents a single polypeptide. Polypeptides can bedetected by dyes, as here, or by other techniques such asautoradiography. Each polypeptide is characterized by itsisoelectric point (pI) and molecular weight. [Part (b) courtesy ofJ. Celis.]sions more easily than can larger proteins, they travel througha gel filtration column more slowly than do larger proteins(Figure 3-34a). (In contrast, proteins migrate through thepores in an electrophoretic gel; thus smaller proteins movefaster than larger ones.) The total volume of liquid requiredto elute a protein from a gel filtration column depends on itsmass: the smaller the mass, the greater the elution volume.
Byuse of proteins of known mass, the elution volume can be usedto estimate the mass of a protein in a mixture.Ion-Exchange Chromatography In a second type of liquidchromatography, called ion-exchange chromatography, proteins are separated on the basis of differences in theircharges. This technique makes use of specially modifiedbeads whose surfaces are covered by amino groups or carboxyl groups and thus carry either a positive charge (NH3)or a negative charge (COO) at neutral pH.The proteins in a mixture carry various net charges atany given pH.
When a solution of a protein mixture flowsthrough a column of positively charged beads, only proteinswith a net negative charge (acidic proteins) adhere to thebeads; neutral and positively charged (basic) proteins flowunimpeded through the column (Figure 3-34b). The acidicproteins are then eluted selectively by passing a gradient ofincreasing concentrations of salt through the column.
At low913.6 • Purifying, Detecting, and Characterizing Proteins(c) Antibody-affinity chromatography(a) Gel filtration chromatographyLoad inpH 7 bufferLarge proteinSmall proteinLayersampleoncolumnAdd bufferto washproteinsthroughcolumnPolymer gel beadCollectfractions321Proteinrecognizedby antibodyElutewithpH 3bufferWashProtein notrecognizedby antibodyAntibody321(b) Ion-exchange chromatographyNegatively chargedproteinPositively chargedproteinLayersampleoncolumnCollectpositivelychargedproteinsElute negativelycharged proteinwith salt solution(NaCl)Na+Positively charged gel beadCl−4321▲ EXPERIMENTAL FIGURE 3-34 Three commonly usedliquid chromatographic techniques separate proteins on thebasis of mass, charge, or affinity for a specific ligand.
(a) Gelfiltration chromatography separates proteins that differ in size.A mixture of proteins is carefully layered on the top of a glasscylinder packed with porous beads. Smaller proteins travelthrough the column more slowly than larger proteins. Thusdifferent proteins have different elution volumes and can becollected in separate liquid fractions from the bottom. (b) Ionexchange chromatography separates proteins that differ in netcharge in columns packed with special beads that carry either apositive charge (shown here) or a negative charge. Proteinshaving the same net charge as the beads are repelled and flowthrough the column, whereas proteins having the oppositecharge bind to the beads. Bound proteins—in this case,negatively charged—are eluted by passing a salt gradient (usuallyof NaCl or KCl) through the column. As the ions bind to thebeads, they desorb the protein.
(c) In antibody-affinitychromatography, a specific antibody is covalently attached tobeads packed in a column. Only protein with high affinity for theantibody is retained by the column; all the nonbinding proteinsflow through. The bound protein is eluted with an acidic solution,which disrupts the antigen–antibody complexes.salt concentrations, protein molecules and beads are attracted by their opposite charges.
At higher salt concentrations, negative salt ions bind to the positively charged beads,displacing the negatively charged proteins. In a gradient ofincreasing salt concentration, weakly charged proteins areeluted first and highly charged proteins are eluted last. Similarly, a negatively charged column can be used to retain andfractionate basic proteins.92CHAPTER 3 • Protein Structure and FunctionAffinity Chromatography The ability of proteins to bindspecifically to other molecules is the basis of affinity chromatography. In this technique, ligand molecules that bind tothe protein of interest are covalently attached to the beadsused to form the column.
Ligands can be enzyme substratesor other small molecules that bind to specific proteins. In awidely used form of this technique, antibody-affinity chromatography, the attached ligand is an antibody specific forthe desired protein (Figure 3-34c).An affinity column will retain only those proteins thatbind the ligand attached to the beads; the remaining proteins, regardless of their charges or masses, will passthrough the column without binding to it. However, if a retained protein interacts with other molecules, forming acomplex, then the entire complex is retained on the column.The proteins bound to the affinity column are then eluted byadding an excess of ligand or by changing the salt concentration or pH.
The ability of this technique to separate particular proteins depends on the selection of appropriateligands.Highly Specific Enzyme and Antibody AssaysCan Detect Individual ProteinsThe purification of a protein, or any other molecule, requiresa specific assay that can detect the molecule of interest in column fractions or gel bands. An assay capitalizes on somehighly distinctive characteristic of a protein: the ability tobind a particular ligand, to catalyze a particular reaction, orto be recognized by a specific antibody.
An assay must alsobe simple and fast to minimize errors and the possibility thatthe protein of interest becomes denatured or degraded whilethe assay is performed. The goal of any purification schemeis to isolate sufficient amounts of a given protein for study;thus a useful assay must also be sensitive enough that only asmall proportion of the available material is consumed.Many common protein assays require just from 109 to1012 g of material.Chromogenic and Light-Emitting Enzyme Reactions Manyassays are tailored to detect some functional aspect of a protein. For example, enzyme assays are based on the ability todetect the loss of substrate or the formation of product.Some enzyme assays utilize chromogenic substrates, whichchange color in the course of the reaction.
(Some substratesare naturally chromogenic; if they are not, they can be linkedto a chromogenic molecule.) Because of the specificity of anenzyme for its substrate, only samples that contain the enzyme will change color in the presence of a chromogenic substrate and other required reaction components; the rate ofthe reaction provides a measure of the quantity of enzymepresent.Such chromogenic enzymes can also be fused or chemically linked to an antibody and used to “report” the presenceor location of the antigen.
Alternatively, luciferase, an enzyme present in fireflies and some bacteria, can be linked toan antibody. In the presence of ATP and luciferin, luciferasecatalyzes a light-emitting reaction. In either case, after theantibody binds to the protein of interest, substrates of thelinked enzyme are added and the appearance of color or1 Electrophoresis/transferAntibody detection4 Chromogenic detection23Technique Animation: ImmunoblottingMEDIA CONNECTIONSElectriccurrentSDS-polyacrylamide gelMembraneIncubate withAb1 ( );wash excess▲ EXPERIMENTAL FIGURE 3-35 Western blotting(immunoblotting) combines several techniques to resolveand detect a specific protein. Step 1 : After a proteinmixture has been electrophoresed through an SDS gel, theseparated bands are transferred (blotted) from the gel onto aporous membrane.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
