- Email: [email protected]
High resolution two-dimensional polyacrylamide gel electrophoresis
tmak’in analyricalchemirt~, ml. 2, no. 9, I983
211
High resolution twwdimensional polyacrylamide gel electrophoresis The combination of two different gel eiectrophoretic techniques in a two-dimensional separation procedure provides the resolution capacity required for the simultaneous separation and analysis of complex protein mixtures. This technique is therefore a powerful tool for the study of phenotyplc expression. Michael J. Dunn and Arthur H. M. Burghes London, UK Onedimensional electrophoretic methods cannot resolve the several thousand gene products synthesized by any cell type. Two-dimensional (2D) procedures substantially increase resolution, making analysis of complex protein mixtures a practical possibility. Although the history of 2D electrophoresis can be traced back over 25 years, it was O’Farrell’ who took advantage of important methodological advances and described a technique of PD-polyacrylamide gel electrophoresis (PD-PAGE) using isoelectric focusing (IEF) in cylindrical polyacrylamide gels containing urea and nonionic detergent for the first dimension with sodium dodecyl sulphate (SDS)-PAGE in gradient gels for the second dimension. These two procedures separate proteins according to independent parameters (charge and molecular weight), so that the proteins are distributed across the gel, thereby maximizing resolution. This technique is the basis of PD-PAGE technology as practised today. In this article we will describe recent developments in PD-PAGE. For a detailed account the reader is referred to our recent reviews2s3.
Sample solubilizath Sample solubilization should completely disrupt all protein complexes and aggregates into single polypeptide chains, and their persistence results in ‘artefactual’ spots in 2D maps. Although SDS disrupts most protein interactions, its anionic charge makes it unsuitable for inclusion in IEF gels. The combination of9 M urea and 2% NP-40 recommended by O’Farell’ does not disaggregate all protein complexes. The inclusion of SDS improves solubilization and entry of proteins, but even small amounts of SDS remove all detergent from the IEF gel due to the formation of mixed micelles, exposing the proteins to a detergent-free environment which can result in their precipitation. Protein-nucleic acid interactions can also interfere with solubilization; DNA and RNA removal can therefore be beneficial. Attempts have been made to use alternative detergents and denaturants, but some of these are incompatible with polyacrylamide. Thus, there is a need for better sample solubilization procedures and, perhaps, alternative support matrices for PD-PAGE, 0165-9936/83/$01.00
IEF dimension IEF is an equilibrium technique as proteins have no net charge at their isoelectric point (PI). The approach of any ampholyte to equilibrium is asymptotic and the number of volt hours (Vh) required for a protein to focus can vary with the conditions. Polyacrylamide is a restrictive matrix which can exert a molecular sieving effect. Therefore, weak gels (3-5%) should be used for IEF, but these can still restrict the migration of high molecular weight proteins. In addition, IEF gels containing urea require longer focusing times. For optimal resolution equilibrium conditions can be used. These are determined by the constancy of the separation pattern over long focusing times, or by the coincidence of bands when samples are migrated from the anode and cathode. Useful 2D separations can be obtained under non-equilibrium conditions, but rigorous control of focusing time (Vh) and gel length is required to ensure reproducibility. A major disadvantage of conventional PD-PAGE is severe cathodic drift with the consequent loss of basic proteins from the gels. To improve resolution at basic pH, a procedure for non-equilibrium pH gradient electrophoresis (NEPHGE) has been developed4. Proteins are loaded at the acid end of the gel and electrophoresed for a relatively short time. The proteins are separated in a rapidly forming pH gradient and their distribution is determined by the time of electrophoresis. Unfortunately, both an equilibrium IEF and NEPHGE gel are required for the complete analysis of any sample. Another disadvantage of non-equilibrium systems is that they do not separate proteins purely by their charge properties, with the result that the two parameters for the 2D separation are not independent. We obtained improved resolution of cathodic proteins using IEF gels containing urea and NP-40, cast on silanized glass plates and run on a flat-bed apparatus. We have adapted this method for 2D separations5. The gels are cast on derivatized plastic supports which are stable in the presence of both urea and NP4-O. A gel obtained using this procedure is shown in Fig. lc and is compared with gels obtained using IEF (Fig. lb) and NEPHGE (Fig. la) rod gel techniques. Proteins which must normally be analysed by NEPHGE are well-resolved in an equilibrium system. 0 1983 Elscvicr Science Publishers B.V.
trendc in analyticalchemistry,vol. 2, no. 9, 1983
Fig. 1. Typical 20 separations obtained b (a) O’Farsell IEF, (b) NEPHGE, and (c)Jat-bed IEF 2D-PAGE techniques. [(a) and (b) from Bravo and Celi s (1980). Reprinted by permission from Exp. Cell Res. 127, 249-260. Copyright 1980. Academic Press.]
The quality of the IEF separation depends on the ampholytes forming the pH gradient. As proteins can vary by fractional charges it is essential to maximize the number of ampholytes in the gel. Thus, blending of different carrier ampholytes to give a mixture containing more species should improve the resolution of proteins in IEF. We found that such mixtures substantially improved separation by IEF, resulting in enhanced resolution in the second dimension gel’. It has recently been found that the use of different ampholytes can result in protein spots occupying different positions (i.e. different apparent PI’S) in 2D maps. This phenomenon could be a reflection of protein-ampholyte interactions. Alternatives to synthetic carrier ampholytes have recently been described. A complex mixture of 47 different buffers (‘buffer focusing’) produced increased pH gradient stability’. Another advance has been the
development of ‘Immobilines’ for the formation of immobilized pH gradients (see Righetti, this issue). This technique was originally restricted to narrow pH gradients (0.1 pH units), but has recently been adapted for extended gradients (3-4 pH units) suitable for the first dimension of 2D-PAGE. Immobilized pH gradients offer the advantage of good reproducibility, together with the ability to withstand high voltages and long focusing times without drift.
Transfer between dimensions IEF gels are usually equilibrated before application to the second dimension gel, but diffusion during this procedure can result in loss of protein and band broadening detrimental to resolution. However, very short equilibration times can result in streaking of high molecular weight proteins and retention of sample within the IEF gel.
trmdr‘inaaaIytia1 chcmisiry, vol. 2,
213
no. 9,1983
SDS dimension The first dimension IEF gel is placed on top of the second dimension SDS slab gel and is usually cemented in place using agarose. It is not necessary to use a stacking gel. Resolution of 2D-PAGE depends on the maximum area of the gel being used for separation. However, protein subunit molecular weights are clustered around a mean value, so that neither a gel of a single polyacrylamide concentration nor a linear polyacrylamide gradient will produce a uniform distribution of spots. It is posssible, but somewhat laborious, to run a series of SDS gels of different concentrations. We have described a method of reproducibly casting SDS polyacrylamide gradient gels with a flattened mid-region which spreads proteins in the SDS dimension5. In addition, using ampholyte mixtures it is possible to construct pH gradients with non-linear shapes designed to distribute protein uniformly in the IEF dimension’. Several procedures exist for casting and running multiple batches of gels which help to increase reproducibility. For example, in the ‘Isodalt’ procedure% * 20 IEF and 10 SDS-PAGE gels can be run simultaneously. Such techniques can lead to problems in handling large numbers of SDS slab gels, but it is now possible, using silanes, to bind polyacrylamide to glass or plastic. However, these procedures can be unreliable, and this problem can be overcome only if covalent binding of gel to support can be achieved. An additional advantage of having gels bound to supports is that no stretching of the gel can occur, thereby minimizing pattern distortion.
Resolution capacity of 2D-PAGE A typical mammalian cell expresses about 2 000 proteins at a physiologically significant level at any one time, which would result in 3 000 to 4 000 spots in a 2D separation. Can PD-PAGE resolve this number of components? Based on the resolution of onedimensional IEF and SDS-PAGE techniques (about 100 bands each), PD-PAGE should be able to resolve 10 000 spots’. However, resolution is degraded by factors such as insolubility ofproteins, streakingofhigh molecular weight components, spreading of spots and difficulties in detecting minor components so that, in practice, resolution is in the range of 1 000 to 2 000 spots.
Protein detection Trichloroacetic acid (TCA) is the best fixative available for 2D gels. Recently, silver staining methods reputed to be 100 to 200 times more sensitive than Coomassie Blue have been developed (see Allen, this issue). Radioactive methods can be used to increase detection sensitivity. Proteins can be labelled by incorporation of radiolabelled metabolites during protein synthesis or by post-synthetic labelling procedures, and identified in 2D patterns by autoradiography. Detection sensitivity for low-energy P-emitters can be increased using ‘fluorography’ in
which the gels are impregnated with an organic scintillator. A major problem in the use of PD-PAGE is that variations in reproducibility complicate the comparison of gels. Such comparisons are simplified if the samples are radiolabelled with different isotopes and run on the same 2D gel. However, it must be established that the metabolites labelled with different isotopes are utilized in the same way. The method depends on the ability to distinguish between the two isotopes used. For pairs of PHI- and [“Cl-1abelled samples this can be achieved using fluorography to visualize the weaker isotope, i.e. [aH], while using direct autoradiography to detect the more energetic [‘“cl P-particles. In order to detect only [‘HI &particles by fluorography it is essential to use a high [3H] : [‘“Cl ratio in the sample, but this necessitates prolonged autoradiographic exposures. However, it is possible to use a composite fluorographic image due to both isotopes. If a contact negative of the fluorogram is placed in register with the direct autoradiograph, polypeptides labelled exclusively with [3H] a pear as white spots, while those labelled with both P3H] and [‘“cl, or [‘“Cl alone, appear as black spots or spots with white halos”.
Quantitation and analysis PD-PAGE patterns are almost impossible to analyse by simple visual inspection. This has resulted in the development of computer systems which can quantitatively analyse and match 2D maps’. The essential features of these systems are outlined below. An image of the stained gel or autoradiogram is first captured using either a scanning densitometer (drum or flat-bed type) or a video camera. This image is then subjected to background subtraction and filtering to reduce ‘noise’. The spots must then be detected, but this is complicated as not all spots are resolved as discrete entities, many being contiguous or overlapping. Procedures such as thresholding, chain assembly, central core analysis and convolution have been applied to solve this problem. In some procedures gaussian fitting is used to resolve overlapping spots and determine spot volume. From these data, quantitative estimates of spot protein can be simply obtained. Unfortunately, variability due to non-linear heterogeneities which exist between gels complicates intergel comparisons. Computer routines are thus necessary to bring the images to be compared into registration, a set of characteristic landmark spots being used to normalize the patterns. The tinal requirement is for computerized databases to catalogue the large volume of data generated by experiments involving many 2D gels.
identification and characterization The value of PD-PAGE is increased if functional information concerning the separated proteins can be obtained. Molecular weight and p1 values themselves are of limited value. If purified proteins are available,
214
trendsin analyticalchemistry,uol. 2, no. 9’ 1983
these can be used to identify the components in the protein mixture being investigated. Various specific staining techniques are available, but of these the potentially most powerful are those based on immunological cross-reactivity. This approach has been revolutionized by blotting tech‘niques for transferring proteins from gels onto substrates such as nitrocellulose. In this state the proteins are readily accessible to probes such as specific monoclonal antisera. The polyclonal and immunocomplexes formed can be visualized using a second antibody or protein A labelled with [‘““I], horseradish peroxidase or fluorescein isothiocyanate. It is possible to excise and recover selected protein spots from 2D gels. Proteins isolated in this way can be readily characterized by enzyme analysis, amino acid analysis, peptide mapping, and protein sequencing, providing that suitable microanalytical techniques are available. In addition, monoclonal antibodies can be raised against the recovered proteins.
Conclusion Recent developments in 2D-PAGE make it possible to analyse complex protein mixtures. The technique is therefore a potent research tool in many areas of biological and biomedical research, but problems remain to be resolved and procedures standardized before 2D-PAGE can be considered to be a routine procedure. Nevertheless, the high resolution capacity
Atmlications
of 2D-PAGE for proteins and recent advances in genetic engineering techniques for studies of genome organization now provide methods for detailed analysis of both genotypic and phenotypic expression.
Acknowledgements We gratefully acknowledge financial support from the Muscular Dystrophy Group of Great Britain and the Medical Research Council.
References 1 2 3 4
O’Farrell, Dunn, M. Dunn, M. O’Farrell, 12, 1133 5 Burghes,
P. H. (1975)j. Biol. Chem. 250, 4017 J. and Burghes, A. H. M. (1983) Electrophoresis 4,97 J. and Burghes, A. H. M. (1983) Electrophoresis 4,173 P., Goodman, M. M. and O’Farrell, P. H. (1977) Cell
Electrophoresis
Cuono, C. 7 Anderson, 331 8 Anderson, 341 9 Anderson, Anderson, 10 McConkey, 6
A. H. M., 3,
Dunn,
M. J.
and Dubowitz,
V.
(1982)
354
B. and Chapo, G. A. (1982) Electrophoresis 3, 65 N. L. and Anderson, N. G. (1978) Anal. Biochem. 85, N. L. and Anderson,
N. G. (1978) Anal. Biochem. 85,
N. L., Taylor, J., Scandora, A. E., Coulter, B. P. and N. G. (1981) Clin. Gem. (Winston Salem. NC) 27, 1807 E. H. (1976) Anal. Biochem. 96, 39
Michael J. Dunn and Arthur H. M. Burghes are members of th research staff of theJerry Lewis Muscle Research Centre at the Royal Postgraduate Medical School, Duane Road, London WI2 OHS, UK. Dr Dunn is President of the British Electrophoresis Society.
of electrofocusina
&&focusing is an electrophoretic technique in which y protein or peptide migrates through a pH gradient under the
influence of an electric current until it reaches the pH where its net charge is zero, this is the isoelectric point, pl. Inherent in electrofocusing is a concentrating effect which counteracts diffusion and provides much greater resolution than most other electrophoretic techniques. It has been used to great advantage in the isolation and identification of genetic variants of many proteins. Roger Bishop Bromma, Sweden Since carrier ampholytes and equipment for electrofocusing first became commercially available in 1966, well over 5 000 articles1 and several application reviews2*3 on the technique have been published. To avoid repetition, in this article I would like to describe some of the most recent and novel applications of electrofocusing.
Electrofocusing in biochemistry, for its own sake Estimates of the smallest difference in isoelectric point for two proteins which can just be separated are about 0.01 pH units for carrier-ampholyte based systems (e.g. ‘LKB Ampholine’ carrier ampholytes) and 0.001 pH units for immobilized pH gradients 0165~~36/83/$01.00
the recently-introduced ‘LKB provided by Immobiline’ System. Thus, electrofocusing has been used for the analysis of all kinds of proteins, from acetaldehyde dehydrogenase to zein. Electrofocusing is also widely used in the preparative purification of proteins, because of the combination of high resolution and the concentrating effect, as well as its simplicity of use. Unlike such techniques as gel permeation and ion exchange chromatography, the resolution in electrofocusing does not fall greatly as the amount of sample applied is increased. For example, in our laboratories, 7 ml of serum has been fractionated on a 5 mm-thick preparative Immobiline gel slab with a pH gradient of pH 4-6, whilst retaining the resolution typical of a 0.5 mm-thick analytical polyacrylamide gel (Ek, K., Bjellqvist, B. and Righetti, P. G., unpublished observations). Fig. 1 shows another example of 0 1983 Elsevicr Science Publishers B.V.
211
High resolution twwdimensional polyacrylamide gel electrophoresis The combination of two different gel eiectrophoretic techniques in a two-dimensional separation procedure provides the resolution capacity required for the simultaneous separation and analysis of complex protein mixtures. This technique is therefore a powerful tool for the study of phenotyplc expression. Michael J. Dunn and Arthur H. M. Burghes London, UK Onedimensional electrophoretic methods cannot resolve the several thousand gene products synthesized by any cell type. Two-dimensional (2D) procedures substantially increase resolution, making analysis of complex protein mixtures a practical possibility. Although the history of 2D electrophoresis can be traced back over 25 years, it was O’Farrell’ who took advantage of important methodological advances and described a technique of PD-polyacrylamide gel electrophoresis (PD-PAGE) using isoelectric focusing (IEF) in cylindrical polyacrylamide gels containing urea and nonionic detergent for the first dimension with sodium dodecyl sulphate (SDS)-PAGE in gradient gels for the second dimension. These two procedures separate proteins according to independent parameters (charge and molecular weight), so that the proteins are distributed across the gel, thereby maximizing resolution. This technique is the basis of PD-PAGE technology as practised today. In this article we will describe recent developments in PD-PAGE. For a detailed account the reader is referred to our recent reviews2s3.
Sample solubilizath Sample solubilization should completely disrupt all protein complexes and aggregates into single polypeptide chains, and their persistence results in ‘artefactual’ spots in 2D maps. Although SDS disrupts most protein interactions, its anionic charge makes it unsuitable for inclusion in IEF gels. The combination of9 M urea and 2% NP-40 recommended by O’Farell’ does not disaggregate all protein complexes. The inclusion of SDS improves solubilization and entry of proteins, but even small amounts of SDS remove all detergent from the IEF gel due to the formation of mixed micelles, exposing the proteins to a detergent-free environment which can result in their precipitation. Protein-nucleic acid interactions can also interfere with solubilization; DNA and RNA removal can therefore be beneficial. Attempts have been made to use alternative detergents and denaturants, but some of these are incompatible with polyacrylamide. Thus, there is a need for better sample solubilization procedures and, perhaps, alternative support matrices for PD-PAGE, 0165-9936/83/$01.00
IEF dimension IEF is an equilibrium technique as proteins have no net charge at their isoelectric point (PI). The approach of any ampholyte to equilibrium is asymptotic and the number of volt hours (Vh) required for a protein to focus can vary with the conditions. Polyacrylamide is a restrictive matrix which can exert a molecular sieving effect. Therefore, weak gels (3-5%) should be used for IEF, but these can still restrict the migration of high molecular weight proteins. In addition, IEF gels containing urea require longer focusing times. For optimal resolution equilibrium conditions can be used. These are determined by the constancy of the separation pattern over long focusing times, or by the coincidence of bands when samples are migrated from the anode and cathode. Useful 2D separations can be obtained under non-equilibrium conditions, but rigorous control of focusing time (Vh) and gel length is required to ensure reproducibility. A major disadvantage of conventional PD-PAGE is severe cathodic drift with the consequent loss of basic proteins from the gels. To improve resolution at basic pH, a procedure for non-equilibrium pH gradient electrophoresis (NEPHGE) has been developed4. Proteins are loaded at the acid end of the gel and electrophoresed for a relatively short time. The proteins are separated in a rapidly forming pH gradient and their distribution is determined by the time of electrophoresis. Unfortunately, both an equilibrium IEF and NEPHGE gel are required for the complete analysis of any sample. Another disadvantage of non-equilibrium systems is that they do not separate proteins purely by their charge properties, with the result that the two parameters for the 2D separation are not independent. We obtained improved resolution of cathodic proteins using IEF gels containing urea and NP-40, cast on silanized glass plates and run on a flat-bed apparatus. We have adapted this method for 2D separations5. The gels are cast on derivatized plastic supports which are stable in the presence of both urea and NP4-O. A gel obtained using this procedure is shown in Fig. lc and is compared with gels obtained using IEF (Fig. lb) and NEPHGE (Fig. la) rod gel techniques. Proteins which must normally be analysed by NEPHGE are well-resolved in an equilibrium system. 0 1983 Elscvicr Science Publishers B.V.
trendc in analyticalchemistry,vol. 2, no. 9, 1983
Fig. 1. Typical 20 separations obtained b (a) O’Farsell IEF, (b) NEPHGE, and (c)Jat-bed IEF 2D-PAGE techniques. [(a) and (b) from Bravo and Celi s (1980). Reprinted by permission from Exp. Cell Res. 127, 249-260. Copyright 1980. Academic Press.]
The quality of the IEF separation depends on the ampholytes forming the pH gradient. As proteins can vary by fractional charges it is essential to maximize the number of ampholytes in the gel. Thus, blending of different carrier ampholytes to give a mixture containing more species should improve the resolution of proteins in IEF. We found that such mixtures substantially improved separation by IEF, resulting in enhanced resolution in the second dimension gel’. It has recently been found that the use of different ampholytes can result in protein spots occupying different positions (i.e. different apparent PI’S) in 2D maps. This phenomenon could be a reflection of protein-ampholyte interactions. Alternatives to synthetic carrier ampholytes have recently been described. A complex mixture of 47 different buffers (‘buffer focusing’) produced increased pH gradient stability’. Another advance has been the
development of ‘Immobilines’ for the formation of immobilized pH gradients (see Righetti, this issue). This technique was originally restricted to narrow pH gradients (0.1 pH units), but has recently been adapted for extended gradients (3-4 pH units) suitable for the first dimension of 2D-PAGE. Immobilized pH gradients offer the advantage of good reproducibility, together with the ability to withstand high voltages and long focusing times without drift.
Transfer between dimensions IEF gels are usually equilibrated before application to the second dimension gel, but diffusion during this procedure can result in loss of protein and band broadening detrimental to resolution. However, very short equilibration times can result in streaking of high molecular weight proteins and retention of sample within the IEF gel.
trmdr‘inaaaIytia1 chcmisiry, vol. 2,
213
no. 9,1983
SDS dimension The first dimension IEF gel is placed on top of the second dimension SDS slab gel and is usually cemented in place using agarose. It is not necessary to use a stacking gel. Resolution of 2D-PAGE depends on the maximum area of the gel being used for separation. However, protein subunit molecular weights are clustered around a mean value, so that neither a gel of a single polyacrylamide concentration nor a linear polyacrylamide gradient will produce a uniform distribution of spots. It is posssible, but somewhat laborious, to run a series of SDS gels of different concentrations. We have described a method of reproducibly casting SDS polyacrylamide gradient gels with a flattened mid-region which spreads proteins in the SDS dimension5. In addition, using ampholyte mixtures it is possible to construct pH gradients with non-linear shapes designed to distribute protein uniformly in the IEF dimension’. Several procedures exist for casting and running multiple batches of gels which help to increase reproducibility. For example, in the ‘Isodalt’ procedure% * 20 IEF and 10 SDS-PAGE gels can be run simultaneously. Such techniques can lead to problems in handling large numbers of SDS slab gels, but it is now possible, using silanes, to bind polyacrylamide to glass or plastic. However, these procedures can be unreliable, and this problem can be overcome only if covalent binding of gel to support can be achieved. An additional advantage of having gels bound to supports is that no stretching of the gel can occur, thereby minimizing pattern distortion.
Resolution capacity of 2D-PAGE A typical mammalian cell expresses about 2 000 proteins at a physiologically significant level at any one time, which would result in 3 000 to 4 000 spots in a 2D separation. Can PD-PAGE resolve this number of components? Based on the resolution of onedimensional IEF and SDS-PAGE techniques (about 100 bands each), PD-PAGE should be able to resolve 10 000 spots’. However, resolution is degraded by factors such as insolubility ofproteins, streakingofhigh molecular weight components, spreading of spots and difficulties in detecting minor components so that, in practice, resolution is in the range of 1 000 to 2 000 spots.
Protein detection Trichloroacetic acid (TCA) is the best fixative available for 2D gels. Recently, silver staining methods reputed to be 100 to 200 times more sensitive than Coomassie Blue have been developed (see Allen, this issue). Radioactive methods can be used to increase detection sensitivity. Proteins can be labelled by incorporation of radiolabelled metabolites during protein synthesis or by post-synthetic labelling procedures, and identified in 2D patterns by autoradiography. Detection sensitivity for low-energy P-emitters can be increased using ‘fluorography’ in
which the gels are impregnated with an organic scintillator. A major problem in the use of PD-PAGE is that variations in reproducibility complicate the comparison of gels. Such comparisons are simplified if the samples are radiolabelled with different isotopes and run on the same 2D gel. However, it must be established that the metabolites labelled with different isotopes are utilized in the same way. The method depends on the ability to distinguish between the two isotopes used. For pairs of PHI- and [“Cl-1abelled samples this can be achieved using fluorography to visualize the weaker isotope, i.e. [aH], while using direct autoradiography to detect the more energetic [‘“cl P-particles. In order to detect only [‘HI &particles by fluorography it is essential to use a high [3H] : [‘“Cl ratio in the sample, but this necessitates prolonged autoradiographic exposures. However, it is possible to use a composite fluorographic image due to both isotopes. If a contact negative of the fluorogram is placed in register with the direct autoradiograph, polypeptides labelled exclusively with [3H] a pear as white spots, while those labelled with both P3H] and [‘“cl, or [‘“Cl alone, appear as black spots or spots with white halos”.
Quantitation and analysis PD-PAGE patterns are almost impossible to analyse by simple visual inspection. This has resulted in the development of computer systems which can quantitatively analyse and match 2D maps’. The essential features of these systems are outlined below. An image of the stained gel or autoradiogram is first captured using either a scanning densitometer (drum or flat-bed type) or a video camera. This image is then subjected to background subtraction and filtering to reduce ‘noise’. The spots must then be detected, but this is complicated as not all spots are resolved as discrete entities, many being contiguous or overlapping. Procedures such as thresholding, chain assembly, central core analysis and convolution have been applied to solve this problem. In some procedures gaussian fitting is used to resolve overlapping spots and determine spot volume. From these data, quantitative estimates of spot protein can be simply obtained. Unfortunately, variability due to non-linear heterogeneities which exist between gels complicates intergel comparisons. Computer routines are thus necessary to bring the images to be compared into registration, a set of characteristic landmark spots being used to normalize the patterns. The tinal requirement is for computerized databases to catalogue the large volume of data generated by experiments involving many 2D gels.
identification and characterization The value of PD-PAGE is increased if functional information concerning the separated proteins can be obtained. Molecular weight and p1 values themselves are of limited value. If purified proteins are available,
214
trendsin analyticalchemistry,uol. 2, no. 9’ 1983
these can be used to identify the components in the protein mixture being investigated. Various specific staining techniques are available, but of these the potentially most powerful are those based on immunological cross-reactivity. This approach has been revolutionized by blotting tech‘niques for transferring proteins from gels onto substrates such as nitrocellulose. In this state the proteins are readily accessible to probes such as specific monoclonal antisera. The polyclonal and immunocomplexes formed can be visualized using a second antibody or protein A labelled with [‘““I], horseradish peroxidase or fluorescein isothiocyanate. It is possible to excise and recover selected protein spots from 2D gels. Proteins isolated in this way can be readily characterized by enzyme analysis, amino acid analysis, peptide mapping, and protein sequencing, providing that suitable microanalytical techniques are available. In addition, monoclonal antibodies can be raised against the recovered proteins.
Conclusion Recent developments in 2D-PAGE make it possible to analyse complex protein mixtures. The technique is therefore a potent research tool in many areas of biological and biomedical research, but problems remain to be resolved and procedures standardized before 2D-PAGE can be considered to be a routine procedure. Nevertheless, the high resolution capacity
Atmlications
of 2D-PAGE for proteins and recent advances in genetic engineering techniques for studies of genome organization now provide methods for detailed analysis of both genotypic and phenotypic expression.
Acknowledgements We gratefully acknowledge financial support from the Muscular Dystrophy Group of Great Britain and the Medical Research Council.
References 1 2 3 4
O’Farrell, Dunn, M. Dunn, M. O’Farrell, 12, 1133 5 Burghes,
P. H. (1975)j. Biol. Chem. 250, 4017 J. and Burghes, A. H. M. (1983) Electrophoresis 4,97 J. and Burghes, A. H. M. (1983) Electrophoresis 4,173 P., Goodman, M. M. and O’Farrell, P. H. (1977) Cell
Electrophoresis
Cuono, C. 7 Anderson, 331 8 Anderson, 341 9 Anderson, Anderson, 10 McConkey, 6
A. H. M., 3,
Dunn,
M. J.
and Dubowitz,
V.
(1982)
354
B. and Chapo, G. A. (1982) Electrophoresis 3, 65 N. L. and Anderson, N. G. (1978) Anal. Biochem. 85, N. L. and Anderson,
N. G. (1978) Anal. Biochem. 85,
N. L., Taylor, J., Scandora, A. E., Coulter, B. P. and N. G. (1981) Clin. Gem. (Winston Salem. NC) 27, 1807 E. H. (1976) Anal. Biochem. 96, 39
Michael J. Dunn and Arthur H. M. Burghes are members of th research staff of theJerry Lewis Muscle Research Centre at the Royal Postgraduate Medical School, Duane Road, London WI2 OHS, UK. Dr Dunn is President of the British Electrophoresis Society.
of electrofocusina
&&focusing is an electrophoretic technique in which y protein or peptide migrates through a pH gradient under the
influence of an electric current until it reaches the pH where its net charge is zero, this is the isoelectric point, pl. Inherent in electrofocusing is a concentrating effect which counteracts diffusion and provides much greater resolution than most other electrophoretic techniques. It has been used to great advantage in the isolation and identification of genetic variants of many proteins. Roger Bishop Bromma, Sweden Since carrier ampholytes and equipment for electrofocusing first became commercially available in 1966, well over 5 000 articles1 and several application reviews2*3 on the technique have been published. To avoid repetition, in this article I would like to describe some of the most recent and novel applications of electrofocusing.
Electrofocusing in biochemistry, for its own sake Estimates of the smallest difference in isoelectric point for two proteins which can just be separated are about 0.01 pH units for carrier-ampholyte based systems (e.g. ‘LKB Ampholine’ carrier ampholytes) and 0.001 pH units for immobilized pH gradients 0165~~36/83/$01.00
the recently-introduced ‘LKB provided by Immobiline’ System. Thus, electrofocusing has been used for the analysis of all kinds of proteins, from acetaldehyde dehydrogenase to zein. Electrofocusing is also widely used in the preparative purification of proteins, because of the combination of high resolution and the concentrating effect, as well as its simplicity of use. Unlike such techniques as gel permeation and ion exchange chromatography, the resolution in electrofocusing does not fall greatly as the amount of sample applied is increased. For example, in our laboratories, 7 ml of serum has been fractionated on a 5 mm-thick preparative Immobiline gel slab with a pH gradient of pH 4-6, whilst retaining the resolution typical of a 0.5 mm-thick analytical polyacrylamide gel (Ek, K., Bjellqvist, B. and Righetti, P. G., unpublished observations). Fig. 1 shows another example of 0 1983 Elsevicr Science Publishers B.V.