H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 39
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Step 2 : The membrane is flooded with asolution of antibody (Ab1) specific for the desired protein.Only the band containing this protein binds the antibody,forming a layer of antibody molecules (although their positionIncubate with enzymelinked Ab2 ( );wash excessReact with substratefor Ab2-linked enzymecannot be seen at this point). After sufficient time for binding,the membrane is washed to remove unbound Ab1. Step 3 :The membrane is incubated with a second antibody (Ab2) thatbinds to the bound Ab1. This second antibody is covalentlylinked to alkaline phosphatase, which catalyzes a chromogenicreaction.
Step 4 : Finally, the substrate is added and a deeppurple precipitate forms, marking the band containing thedesired protein.3.6 • Purifying, Detecting, and Characterizing Proteinsemitted light is monitored. A variation of this technique, particularly useful in detecting specific proteins within livingcells, makes use of green fluorescent protein (GFP), a naturally fluorescent protein found in jellyfish (see Figure 5-46).Western Blotting A powerful method for detecting a particular protein in a complex mixture combines the superiorresolving power of gel electrophoresis, the specificity of antibodies, and the sensitivity of enzyme assays. Called Westernblotting, or immunoblotting, this multistep procedure iscommonly used to separate proteins and then identify a specific protein of interest. As shown in Figure 3-35, two different antibodies are used in this method, one specific for thedesired protein and the other linked to a reporter enzyme.Radioisotopes Are Indispensable Toolsfor Detecting Biological MoleculesA sensitive method for tracking a protein or other biological molecule is by detecting the radioactivity emitted from radioisotopes introduced into the molecule.
At least one atomin a radiolabeled molecule is present in a radioactive form,called a radioisotope.Radioisotopes Useful in Biological Research Hundreds ofbiological compounds (e.g., amino acids, nucleosides, andnumerous metabolic intermediates) labeled with various radioisotopes are commercially available.
These preparationsvary considerably in their specific activity, which is theamount of radioactivity per unit of material, measured in disintegrations per minute (dpm) per millimole. The specific activity of a labeled compound depends on the probability ofdecay of the radioisotope, indicated by its half-life, which isthe time required for half the atoms to undergo radioactivedecay. In general, the shorter the half-life of a radioisotope,the higher its specific activity (Table 3-3).The specific activity of a labeled compound must be highenough that sufficient radioactivity is incorporated into cellular molecules to be accurately detected.
For example, methionine and cysteine labeled with sulfur-35 (35S) are widelyused to label cellular proteins because preparations of these`TABLE 3-3Radioisotopes Commonly Usedin Biological ResearchIsotopeHalf-LifePhosphorus-3214.3 daysIodine-12560.4 daysSulfur-3587.5 daysTritium (hydrogen-3)12.4 yearsCarbon-145730.4 years93amino acids with high specific activities (>1015 dpm/mmol)are available. Likewise, commercial preparations of 3Hlabeled nucleic acid precursors have much higher specificactivities than those of the corresponding 14C-labeled preparations.
In most experiments, the former are preferable because they allow RNA or DNA to be adequately labeled aftera shorter time of incorporation or require a smaller cell sample. Various phosphate-containing compounds in whichevery phosphorus atom is the radioisotope phosphorus-32are readily available. Because of their high specific activity,32P-labeled nucleotides are routinely used to label nucleicacids in cell-free systems.Labeled compounds in which a radioisotope replacesatoms normally present in the molecule have the same chemical properties as the corresponding nonlabeled compounds.Enzymes, for instance, cannot distinguish between substrateslabeled in this way and their nonlabeled substrates.
In contrast, labeling with the radioisotope iodine-125 (125I) requires the covalent addition of 125I to a protein or nucleicacid. Because this labeling procedure modifies the chemicalstructure of a protein or nucleic acid, the biological activityof the labeled molecule may differ somewhat from that of thenonlabeled form.Labeling Experiments and Detection of RadiolabeledMolecules Whether labeled compounds are detected by autoradiography, a semiquantitative visual assay, or their radioactivity is measured in an appropriate “counter,” a highlyquantitative assay that can determine the concentration of aradiolabeled compound in a sample, depends on the natureof the experiment.
In some experiments, both types of detection are used.In one use of autoradiography, a cell or cell constituentis labeled with a radioactive compound and then overlaidwith a photographic emulsion sensitive to radiation. Development of the emulsion yields small silver grains whose distribution corresponds to that of the radioactive material.Autoradiographic studies of whole cells were crucial in determining the intracellular sites where various macromolecules are synthesized and the subsequent movements of thesemacromolecules within cells.
Various techniques employingfluorescent microscopy, which we describe in the next chapter, have largely supplanted autoradiography for studies ofthis type. However, autoradiography is commonly used invarious assays for detecting specific isolated DNA or RNAsequences (Chapter 9).Quantitative measurements of the amount of radioactivity in a labeled material are performed with several differentinstruments. A Geiger counter measures ions produced in agas by the particles or rays emitted from a radioisotope.In a scintillation counter, a radiolabeled sample is mixed witha liquid containing a fluorescent compound that emits a flashof light when it absorbs the energy of the particles or raysreleased in the decay of the radioisotope; a phototube in theinstrument detects and counts these light flashes.
Phosphorimagers are used to detect radiolabeled compounds on a surface, storing digital data on the number of decays inCHAPTER 3 • Protein Structure and FunctionERGolgiSecretorygranulePulseT = 0;add 3H-leucineChaseT = 5 min;wash out 3H-leucineT = 10 minT = 45 min▲ EXPERIMENTAL FIGURE 3-36 Pulse-chase experimentscan track the pathway of protein movement within cells.To determine the pathway traversed by secreted proteinssubsequent to their synthesis on the rough endoplasmicreticulum (ER), cells are briefly incubated in a medium containinga radiolabeled amino acid (e.g., [3H]leucine), the pulse, which willlabel any protein synthesized during this period. The cells arethen washed with buffer to remove the pulse and transferred tomedium lacking a radioactive precursor, the chase. Samplestaken periodically are analyzed by autoradiography to determinethe cellular location of labeled protein.
At the beginning of theexperiment (t 0), no protein is labeled, as indicated by thegreen dotted lines. At the end of the pulse (t 5 minutes), allthe labeled protein (red lines) appears in the ER. At subsequenttimes, this newly synthesized labeled protein is visualized firstin the Golgi complex and then in secretory vesicles. Becauseany protein synthesized during the chase period is not labeled,the movement of the labeled protein can be defined quiteprecisely.disintegrations per minute per small pixel of surface area.These instruments, which can be thought of as a kind ofreusable electronic film, are commonly used to quantitate radioactive molecules separated by gel electrophoresis and arereplacing photographic emulsions for this purpose.A combination of labeling and biochemical techniquesand of visual and quantitative detection methods is often employed in labeling experiments.
For instance, to identify themajor proteins synthesized by a particular cell type, a sampleof the cells is incubated with a radioactive amino acid (e.g.,[35S]methionine) for a few minutes. The mixture of cellularproteins is then resolved by gel electrophoresis, and the gelis subjected to autoradiography or phosphorimager analysis.The radioactive bands correspond to newly synthesized proteins, which have incorporated the radiolabeled amino acid.Alternatively, the proteins can be resolved by liquid chromatography, and the radioactivity in the eluted fractions canbe determined quantitatively with a counter.Pulse-chase experiments are particularly useful for tracing changes in the intracellular location of proteins or thetransformation of a metabolite into others over time.
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