Showing papers in "Methods of Molecular Biology in 1984"

SDS Polyacrylamide Gel Electrophoresis of Proteins.

Abstract: Probably the most widely used of techniques for analyzing mixtures of proteins is SDS polyacrylamide gel electrophoresis. In this technique, proteins are reacted with the anionic detergent, sodium dodecylsulfate (SDS, or sodium lauryl sulfate) to form negatively charged complexes. The amount of SDS bound by a protein, and so the charge on the complex, is roughly proportional to its size. Commonly, about 1.4 g SDS is bound per 1 g protein, although there are exceptions to this rule. The proteins are generally denatured and solubilized by their binding of SDS, and the complex forms a prolate elipsoid or rod of a length roughly proportionate to the protein's molecular weight. Thus, proteins of either acidic or basic pI form negatively charged complexes that can be separated on the bases of differences in charges and sizes by electrophoresis through a sieve-like matr ix of polyacrylamide gel.

The Isolation of High Molecular Weight Eukaryotic DNA

Abstract: The isolation of high molecular weight eukaryotic DNA in good yield is an important prerequisite for the analysis of specific sequences by Southern blotting ( Chapter 9 ), or for molecular cloning in phage or cosmid vectors ( Chapter 49 ).

Enzyme-linked immunosorbant assay (ELISA).

Abstract: In general, immunological methods are not very well suited for a quantitative determination of the antigen to be studied. The ELISA technique, however, can be used for a quantitative or at least semiquantitative determination of the concentration of a certain antigen. The method was first introduced by Engvall and Perlmann (1). The principle of ELISA (see Fig. 1), also called the double antibody sandwich technique, is the following: Antibodies against the antigen to be measured are adsorbed to a solid support, in most cases a polystyrene microtiter plate. After coating the support with antibody and washing, the antigen is added and will bind to the adsorbed antibodies. Next, a conjugate that will also bind to the antigen is added. Conjugates are antibody molecules to which an enzyme is covalently bound. Fig. 1. The main steps in a (noncompetitive) ELISA test. (1) The antibody to the antigen being quantitated is adsorbed onto a solid phase, usually polystyrene. (2) The sample containing the antigen being measured is then added. (3) Following the incubation and washing steps, a second enzyme-labeled antibody is then added. After further incubation and washing steps, enzyme substrate is added. (A substrate is chosen that will give a colored product). The amount of color produced is therefore proportional to the amount of antigen bound to the original antibody.

Gradient SDS Polyacrylamide Gel Electrophoresis

Abstract: The preparation of fixed-concentration polyacrylamide gels has been described in Chapter 6. However, the use of polyacrylamide gels that have a gradient of increasing acrylamide concentration (and hence decreasing pore size) can sometimes have advantages over fixed-concentration acrylamide gels. During electrophoresis in gradient gels, proteins migrate until the decreasing pore size impedes further progress. Once the "pore limit" is reached, the protein banding pattern does not change appreciably with time, although migration does not cease completely. There are three main advantages of gradient gels over linear gels: 1.The advancing edge of the migrating protein zone is retarded more than the trailing edge, thus resulting in a sharpening of the protein bands.2.The gradient in pore size increases the range of molecular weights that can be fractionated in a single gel run.3.Proteins with close molecular weight values are more likely to separate in a gradient gel than a linear gel.

RNA extraction by the guanidine thiocyanate procedure.

Abstract: This method relies on the strong chaotropic nature of the reagents involved to completely denature any ribonuclease (RNase) present in the sample. After lysis in guanidine thiocyanate buffers there are two possibilities for isolation of the RNA. One method involves a series of differential precipitation steps in guanidine hydrochloride (1). The alternative, detailed here, involves centrifugation of the samples on a cushion of 5.7M CsCl (2,3). The RNA passes through this cushion, whereas the DNA and the majority of other cellular macromolecules remain above the cushion.

Agarose gel electrophoresis of DNA.

Abstract: This book contains many chapters describing methods for isolating and modifying DNA molecules. The most usual way of checking the success of such procedures is by looking at the products using electrophoresis in agarose gels. This process separates DNA molecules by size, and the molecules are made visible using the fluorescent dye ethidium bromide. In this way DNA can be checked for size, intactness, homogeneity, and purity. The method is rapid and simple, yet capable of high resolution, and is so sensitive that usually little of the sample is needed for analysis.

Transfer techniques in protein blotting.

Abstract: Polyacrylamide gel electrophoresis is an extremely powerful tool for the analysis of complex protein mixtures. Although the value of this method cannot be questioned, it is restricted in that the separated proteins remain buried within the dense gel matrix and are not readily available for further investigation. A number of methods have been developed in order to try and overcome this problem, for example the elution of proteins from excised gel slices (see Chapter 19 ). Alternatively, proteins have been studied while they are still buried within the gel using a variety of in situ peptide mapping (see Chapter 22 ) and gel overlay techniques (for example, see ref. 1). Unfortunately all of these methods have serious drawbacks: in the case of protein elution and in situ peptide mapping techniques, the resolution and number of bands that can be processed is restricted, whereas the gel overlay techniques are generally time-consuming and insensitive.

Acetic acid-urea polyacrylamide gel electrophoresis of proteins.

Abstract: In SDS polyacrylamide gel electrophoresis, proteins are separated essentially on the basis of their sizes, by the sieving effect of the polyacrylamide gel matrix (see Chapter 6 ). In the absence of SDS, the proteins would still be subject to the sieving effect of the gel matrix, but their charges would vary according to their amino acid content. This is because the charge on a protein at any particular pH is the sum of the charges prevailing on the side chain groups of its constituent amino acid residues, and the free amino and carboxyl groups at its termini (although these are relatively trivial in anything other than a very small peptide). Thus, in an ionic detergent-free gel electrophoretic system, both the molecular size and charge act as bases for effective protein separation. The pH prevailing in such a system might be anything, but is commonly about pH 3. Since the pK ( a ) values of the side chain carboxyl groups of aspartic and glutamic acids are about 3.8 and 4.2, respectively, even these amino acids will contribute little to the negative charge on a protein at this pH. Thus at pH 3, all proteins are likely to be positively charged and to travel towards the cathode in an electric field.

The burton assay for DNA.

Abstract: The Burton assay for DNA is a colorimetric procedure for measuring the deoxyribose moiety of DNA. It is reasonably specific for deoxyribose, although very high concentrations of ribose (from RNA) or sucrose must be avoided. The method can be used on relatively crude extracts and in other circumstances where direct measurement of ultraviolet absorbance of denatured DNA is not practical. The assay has been widely used.

The production of antisera.

Abstract: Suitable antisera are essential for use in all immunochemical procedures. Three important properties of an antiserum are avidity, specificity, and titer. The avidity of an antiserum is a measure of the strength of the interactions of its antibodies with an antigen. The specificity of an antiserum is a measure of the ability of its antibodies to distinguish the immunogen from related antigens. The titer of an antiserum is the final (optimal) dilution at which it is employed in the procedure; it depends on the concentrations of the antibodies present and on their affinities for the antigen. The values of those parameters required for a particular antiserum very much depend on the usage to which the antiserum will be put. For example, for use in radioimmunoassay, it is best to have a monospecific antiserum of high avidity, whereas for use in immunoaffinity chromatography the monospecific antiserum should not possess too high an avidity otherwise it may prove impossible to elute the desired antigen without extensive denaturation.

The purification of poly(a)-containing RNA by affinity chromatography.

Abstract: The vast majority of eukaryotic mRNA molecules contain tracts of poly(adenylic) acid, up to 250 bases in length, at the 3' end. This property is very useful from the point of view of mRNA extraction because it forms the basis of a convenient and simple affinity chromatography procedure (1). Under high salt conditions (0.3-0.5M NaCl or KCl), poly(A) will hybridize to oligo(dT)-cellulose or poly(U)-Sepharose. These commercially available materials consist of polymers of about 10-20 nucleotides, covalently bound to a carbohydrate support, and bind RNA containing a poly(A) tract as short as 20 residues. Ribosomal and transfer RNAs do not possess poly(A) sequences and will not bind (see Note 1).

Preparation of Chromosomal DNA from E. coli.

Abstract: This chapter describes a simple and rapid way of extracting and purifying chromosomal DNA from E. coli and many other species of bacteria. This procedure is essentially a simplified version of that described by Marmur in 1961 (1). The cells are lysed by treatment with a detergent and the mixture is deproteinized by phenol-chloroform extraction. Further purification can be achieved by treatment with ribonuclease and proteinase K. The resulting DNA, free of protein and RNA contamination, is sufficiently pure to be used for restriction digestion and cloning, e.g., in the preparation of gene libraries.

Microsequencing of Peptides and Proteins with 4- N,N -Dimethylaminoazobenzene-4′-isothiocyanate

Abstract: Manual methods for the stepwise N-terminal degradation of polypeptides have been widely applied in protein chemistry. The technique most commonly used up to now, has been the Edman degradation, carried out either manually (1) or automatically (2-4). Although nowadays the reaction can be performed with high efficiency and automatically in a sequencer, the manual methods are still of value. The reasons for this are: (i) The manual methods can be easily applied in any laboratory, even by an inexperienced researcher, with a minimum of equipment at low cost; (ii) It is possible to simultaneously screen many pep-tides for purity, and to gain information in a reasonable time about the N-terminal sequences of sets of peptides generated by proteolytic or chemical cleavage; (iii) The selection of fragments of high purity for the sequencer or those peptides that need to be sequenced in a machine because of length, hydrophobicity, or difficult sequence stretches is made easier.

Fluorography of Polyacrylamide Gels Containing Tritium

Abstract: Fluorography is the term used for the process of determining radioactivity in gels and other media by a combination of fluorescence and photography. Since most of the radiation of a low energy emitter will largely be absorbed by the gel, in the technique of fluorography a fluor (e.g., PPO) is infiltrated into the gel where it can absorb the radiation and re-emit light that will pass through the gel to the film. The resulting photographic image is analogous to an autoradiograph, but for a low energy beta-emitting isotope like (3)H, the sensitivity of fluorography is many times the sensitivity of autoradiography. The fluorograph may be used directly, as a qualitative picture of the radioactivity on the gel. It may also be used to locate radioactive bands or spots that can then be cut from the original gel for further analysis, or be scanned to give quantitative information about the distribution of radioactivity. Figure 1 shows an example of a gel that was stained with Coomassie blue and then fluorographed. Notice that there is no loss of resolution in the fluorography of thin gels of normal size. Fig. 1. This shows two examples of fluorography of protein bands labeled with (3)H. Basic nuclear proteins were isolated from the slime mold, Physarum polycephalum, pulse-labeled with (3)H-acetate in either S phase or G(2) phase of the naturally synchronous cell cycle. The proteins were analyzed by acrylamide gel electrophoresis in acetic acid, urea, and Triton X-100. After electrophoresis, the gel was stained with Coomassie blue, photographed, and then fluorographed. Individual lanes of the gel image were cut from the photograph (negative) and the fluorograph and then printed side-by-side to give the figure shown. Notice that the stain patterns of the two lanes are practically identical, except for the loading, while the radioactivity patterns show major differences, for example, the absence of label in his-tones H2A and H2B in G2 phase (4).

Ligation of DNA with T 4 DNA Ligase

Abstract: Since they are involved in such important processes as DNA replication, DNA repair, and DNA recombination, DNA ligases can be found in all living cells. Two prokaryotic DNA ligases have become indispensible tools in the fields of in vitro DNA recombination and DNA synthesis. DNA ligase from E. coli is a polypeptide with a molecular weight of 74,000 and is NAD-dependent. T(4) DNA ligase is the product of gene 30 of the T(4) phage, has a molecular weight of 68,000, and is ATP-dependent. Both enzymes catalyse the synthesis of a phosphodiester bond between the 3'-hydroxyl group and the 5'-phosphoryl group at a nick in double-stranded DNA. Of the two enzymes, only T(4) DNA ligase is able to join both DNA fragments with protruding ("sticky ends") as well as DNA fragments with "blunt ends" (1). The reaction takes place in three steps. The first step is the transfer of an adenylyl group of ATP (for T(4) ligase) or NAD (for E. coli ligase) to the free enzyme. It is a side chain NH2 group of a lysine residue that becomes adenylated. During this step, pyrophosphate (T(4) enzyme) or nicotinamide monophosphate (E. coli enzyme) are released.In the second step, the adenylyl group is then transferred to the 5'-phosphoryl end of the DNA and in the third step a phosphodiester bond is formed between a 3'-hydroxyl group and the 5'-adenylated phosphoryl group with the release of AMP. [See also ref. (5) for more detail.].

The extraction and isolation of DNA from gels.

Abstract: As will be evident from a number of the following chapters (i.e., Chapters 31 , Chapters 38 - Chapters 41 , Chapters 51 - Chapters 53 ), gel electrophoresis of DNA is a widely used technique in molecular biology. In a number of cases, e.g., for such procedures as cloning and DNA sequencing, it is not sufficient just to analyze the DNA on these gels; the DNA must also be recovered from the gel. It is clear that the DNA in these cases has to be recovered in as high yields as possible and that the molecules should not be damaged. There are many published procedures for extracting DNA fragments from agarose or acrylamide gels (1-4), but none are very satisfactory. As mentioned in Chapters 38 - Chapters 41 , agarose inhibits a number of enzymes used for labeling DNA molecules, for restriction, and for ligation. Acrylamide does not seem to inhibit most enzymes, but interferes with the electron microscopy of DNA. The procedures described below have all been used in our laboratory, albeit with varying degrees of success. The fact that a number of methods have not been included in this chapter does not mean that the particular method could not be of any use, but only that the authors are not familiar with it.The first step in each method is to locate the band of interest, either by staining the DNA with ethidium bromide or, if the DNA is radioactively labeled, by identifying by autoradiography, both of which are described elsewhere in this book and are therefore omitted from this chapter.

The extraction of total RNA by the detergent and phenol method.

Abstract: Successful extraction of RNA depends on the quantitative recovery of pure nucleic acids in an undegraded form. In practice, this means that a selective extraction process is required to remove all the unwanted cellular material in a manner that minimizes degradation of the RNA by hydrolysis or ribonuclease activity. The method described here relies on cell homogenization in an aqueous medium containing a strong detergent (sodium tri-isopropylnaphthalene sulfonate) and a chelating agent (sodium 4-aminosalicylate) to solubilize the cell components. An immiscible solution of phenol is then added to selectively extract hydophobic components and to denature protein. Following phase separation, the RNA is recovered by precipitation from the aqueous phase by the addition of absolute alcohol, thereby separating the RNA from small molecular weight contaminants such as carbohydrates, amino acids, and nucleotides.

Immunodiffusion in gels.

Abstract: Immunodiffusion in gels encompasses a variety of techniques that are useful for the analysis of antigens and antibodies (1). The fundamental immunochemical principles behind their use are exactly the same as those that apply to antigen#x2013;antibody interactions in the liquid state. Thus an antigen will rapidly react its specific antibody to form a complex, the composition of which will depend on the nature, concentrations, and proportions of the initial reactants. As increasing amounts of a multivalent antigen are allowed to react with a fixed amount of antibody, precipitation occurs, in part because of extensive crosslinking between the reactant molecules. Initially the antibody is in excess and all of the added antigen is present in the form of an insoluble antigen#x2013;antibody aggregate. Addition of more antigen leads to the formation of more immune precipitate. However, a point is reached beyond which further addition of antigen produces an excess of antigen and leads to a reduction in the amount of the precipitate (see Fig. 1) because of the formation of soluble antigen#x2013;antibody complexes. Fig. 1. Variation in the amount of immune precipitate on addition of increasing amounts of antigen to a fixed amount of antibody.

High-sensitivity silver staining of proteins following polyacrylamide gel electrophoresis.

Abstract: Polyacrylamide gel electrophoresis is a simple, inexpensive, yet highly versatile and powerful method for the analysis of complex mixtures of proteins. In part, the success of this method has resulted from the ease with which the fractionated proteins can be detected with the blue dye, Coomassie brilliant blue R 250. However, though this stain has proved to be ideal for many of the more traditional applications of this method, it is of limited sensitivity. In particular, the recent development of two-dimensional gel electrophoresis ( Chapter 10 ) and in situ peptide mapping techniques ( Chapter 22 ) have demanded increasingly more sensitive detection methods. In part, this requirement has been met by the use of radioactively labeled proteins, followed by either autoradiography or fluorography ( Chapter 9 ). The principal drawback of this approach is that it involves additional sample preparation and it is often difficult to label the proteins to a sufficiently high specific activity, particularly where proteins are obtained from dilute physiological samples, and so on.

In situ Peptide mapping of proteins following polyacrylamide gel electrophoresis.

Abstract: Polyacrylamide gel electrophoresis is a simple, yet versatile, high resolution technique for the analysis of complex mixtures of proteins. However, this is not to say that this method is without problems. For example, where a series of different protein samples are run on a gel, many of the proteins will have the same mobility and it is frequently impossible to be certain if these bands represent the same protein or whether they simply share similar mobilities. Conversely the samples may contain degradation products or structurally related proteins of differing mobilities.

Carboxy-terminal sequence determination of proteins and peptides with carboxypeptidase y.

Abstract: No useful chemical method (similar to that of the Edman degradation) exists that allows a sequence investigation from the carboxy-terminal end of proteins and peptides. Efforts have therefore been centered around the exploitation of enzymatic methods, notably the use of carboxypeptidases for that purpose. Carboxypeptidases are enzymes that remove amino acids one at a time from the carboxy-terminus of a peptide chain.

The Dansyl Method for Identifying N-Terminal Amino Acids

Abstract: The reagent l-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride, DNS-C1) reacts with the free amino groups of peptides and proteins as shown in Fig. 1. Total acid hydrolysis of the substituted peptide or protein yields a mixture of free amino acids plus the dansyl derivative of the N-terminal amino acid, the bond between the dansyl group and the N-terminal amino acid being resistant to acid hydrolysis. The dansyl amino acid is fluorescent under UV light and is identified by thin-layer chromatography on polyamide sheets. This is an extremely sensitive method for identifying amino acids and in particular has found considerable use in peptide sequence determination when used in conjunction with the Edman degradation (see Chapter 24 ). The dansyl technique was originally introduced by Gray and Hartley (1), and was developed essentially for use with peptides. However, the method can also be applied to proteins (see Note No. 12). Fig. 1. Reaction sequence for the labeling of N-terminal amino acids with dansyl chloride.

Immunoaffinity Purification of Protein Antigens

Abstract: The unique high specificity of antibodies, both polyclonal and monoclonal, makes them extremely valuable tools for rapid, selective purification of antigens. In principle, the antibody immobilized on a column support is used to selectively adsorb antigen from a mixture containing many other proteins (1,2). The other proteins, for which the antibody has no affinity, may be washed away, and the purified antigen then eluted from the immunoadsorbent. In order to dissociate the antigen from its high affinity antibody, the conditions for elution are necessarily extreme (1-3) and thus must be carefully chosen to permit isolation of active protein.

Solid-Phase Screening of Monoclonal Antibodies

Abstract: By far the most convenient and simple assays for screening monoclonal antibody-producing hybridomas are those utilizing solid-phase binding assays, where radiolabelled or enzyme linked antispecies antibodies are used to detect binding of the hybridoma supernatants to insolubilized antigen. Such procedures require an adequate supply of antigen, preferably purified, which can be bound to a solid support, usually through noncovalent binding to PVC microtiter plates or nitrocellulose sheets.

The Use of Restriction Endonucleases

Abstract: The discovery of the mode of action of the class of bacterial enzymes known as restriction endonucleases provided the major breakthrough in opening up the field of genetic engineering. In vivo, these enzymes are involved in recognizing and cutting up foreign DNA entering the cell; their most likely role is thus protecting the bacteria against phage infection. The property that is relevant to us is that these enzymes recognize specific DNA sequences. The enzymes used in DNA manipulations are in fact known as Class II restriction endonucleases; these enzymes cut the DNA within the recognition sequence at a defined point. Treatment of a DNA sample with such enzymes will thus result in each molecule being cut at the same positions and thereby lead to the formation of reproducible fragments.

The Electrophoretic Elution of Proteins from Polyacrylamide Gels

Abstract: The analytical power of acrylamide gel electrophoresis is one of the keys of modern protein chemistry. It is not surprising, therefore, that many methods have been described for converting that analytical power into a preparative tool. None of the available methods is entirely satisfactory for general use since loss of resolution or low recovery is often involved. The method described here has given both high resolution and good recovery but suffers from the disadvantage of being relatively laborious (1,2). In addition, although the recovered proteins are good for peptide analysis or amino acid composition determination, we have found very low yields on Edman degradation of proteins eluted from gels (3).

Radioiodination of proteins.

Abstract: Many different substances can be labeled by radioiodination. Such labeled molecules are of major importance in a variety of investigations, e.g., studies of intermediary metabolism, determinations of agonist and antagonist binding to receptors, quantitative measurements of physiologically active molecules in tissues and biological fluids, and so on. In most of those studies, it is necessary to measure very low concentrations of the particular substance and that in turn implies that it is essential to produce a radioactively labeled tracer molecule of high specific radioactivity. Such tracers, particularly in the case of polypeptides and proteins, can often be conveniently produced by radioiodination.

Manual edman degradation of proteins and peptides.

Abstract: The Edman or phenylisothiocyanate degradation (1) has been employed for the determination of the primary structures of peptides and proteins for approximately three decades. The relative simplicity of the method and its high efficiency in the sequential removal of amino acid residues from the amino terminus of a peptide or protein has resulted in a widespread popularity and usage. In spite of the full automatization of the procedure by Edman and Begg in the nineteen sixties (2), the manual version of the sequential amino acid degradation still remains a very realistic and efficient alternative.

RNA Extraction by the Proteinase K Method

Abstract: This method of RNA extraction relies on a relatively gentle lysis procedure that should burst the cells, but leave the nuclei intact. Contamination of a relatively RNase-free cytoplasmic environment with nuclear nucleases is thus minimised. Next the polysomes are dissociated with SDS and proteinase K and finally the protein is removed by several phenol/choloroform extractions (1,2).

Radiolabeling of DNA by Nick Translation

Abstract: Nick translation is the name given to a reaction that is used to replace cold nucleoside triphosphates in a double-stranded DNA molecule with radioactive ones (1,2). Free 3'-hydroxyl groups are created within the unlabeled DNA (nicks) by deoxyribonuclease 1 (DNAse 1). DNA polymerase 1 from E. coli will then catalyze the addition of a nucleotide residue to the 3'-hydroxyl terminus of the nick. At the same time, the 5'- to 3'-exonuclease activity of this enzyme will eliminate the nucleotide unit from the 5'-phosphoryl terminus of the nick. Thus a new nucleotide with a free 3'-OH group will have been incorporated at the position where the original nucleotide was excised, and the nick will have been shifted along by one nucleotide unit in a 3' direction. This 3' shift, or translation, of the nick will result in the sequential addition of new nucleotides to the DNA while the pre-existing nucleotides will be removed. If radioactively labeled deoxyribonucleoside triphosphates are used as substrates, up to 50% of the residues in the DNA can be labeled.Furthermore, Rigby et al. have shown (2) that the DNA is labeled throughout at a uniform specific activity, which is an important requirement if the DNA is to be used as a probe in molecular hybridization experiments.