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B. S. Dunbar, Two-Dimensional Electrophoresis and Immunological Techniques, Plenum Press, New York, 1987. L. A. Lewis, CRC Handbook of Electrophoresis, CRC Press, Boca Raton, FL, 1980. C. J. Howe and E. S. Ward, Nucleic Acids Sequencing: A Practical Approach, Oxford University Press, New York, 1989. J. Breborowicz and A. Mackiewicz, Eds., Af nity Electrophoresis: Principles and Applications, CRC Press, Boca Raton, FL, 1991. G. H. Keller and M. M. Manak, DNA Probes, Macmillan (Stockton Press), New York, 1989.
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1. D. Rodbard and A. Chrambach, Proc. Natl. Acad. Sci. U.S.A. 65, 1970, 970 977. 2. A. Chrambach, The practice of Quantitative Gel Electrophoresis, VCH Publishers, Deer eld Beach, FL, 1985, p. 177. 3. D. J. Holme and H. Peck, Analytical Biochemistry, 3rd ed., Longman, New York, 1998, pp. 400 402. 4. N. C. Stellwagen, Biochemistry 22, 1983, 6180 6185. 5. D. L. Smisek and D. A. Hoagland, Science 248, 1990, 1221 1223. 6. W. F. Bosron, T.-K. Li, and B. L. Vallee, Biochem. Biophys. Res. Commun. 91, 1979, 1549 1555. 7. W. M. Keung, C. C. Ditlow, and B. L. Vallee, Anal. Biochem. 151, 1985, 92 96. 8. G. H. Keller and M. M. Manak, DNA Probes, Macmillan (Stockton Press), New York, 1993, p. 16. 9. L. T. Kirby, DNA Fingerprinting: An Introduction, Macmillan (Stockton Press), New York, 1990. 10. W. C. Timmer and J. M. Villalobos, J. Chem. Educ. 70, 1993, 273 280. 11. A. M. Maxam and W. Gilbert, Methods Enzymol. 65, 1980, 449 560. 12. R. F. Barker, in Nucleic Acids Sequencing: A Practical Approach, C. J. Howe and E. S. Ward, Eds., Oxford University Press, New York, 1989, p. 133. 13. J. Renart, J. Reiser, and G. R. Stark, Proc. Natl. Acad. Sci. U.S.A. 76, 1979, 3116 3120. 14. K. E. Johansson, in Handbook of Immunoblotting of Proteins, Vol. 1, O. J. Bjerrum and N. H. H. Heegard, Eds., CRC Press, Boca Raton, FL, 1988, pp. 159 165. 15. K. Ogata, in Handbook of Immunoblotting of Proteins, Vol. 1, O. J. Bjerrum and N. H. H. Heegard, Eds., CRC Press, Boca Raton, FL, 1988, pp. 167 176. 16. C. B. Laurell, Anal. Biochem. 15, 1966, 45 52. 17. F. G. Plumley and G. W. Schmidt, Anal. Biochem. 134, 1983, 86 95.
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PROBLEMS 1. The standard and unknown proteins listed below were electrophoresed using SDS PAGE. Estimate the molecular weight of the unknown protein.
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Protein Aldolase Catalase Ferritin Thyroglobulin Unknown MW (kDa) 158 210 440 669 Migration Distance (cm) 3.56 3.23 2.86 2.51 3.03
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2. A protein known to be highly puri ed is subjected to SDS PAGE. Two bands are observed, and these correspond to molecular weights of 50 and 75 kDa. After staining and densitometry, the 75-kDa band is seen to possess twice the area of the 50-kDa band. What conclusions can be reached regarding the original protein
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3. Double-stranded DNA standards and an unknown were electrophoresed in a 0.6% agarose gel, stained with ethidium bromide and photographed under UV light. The negative of the photographed separation pattern was then scanned with a densitometer. The following data were obtained. Estimate the length of the unknown DNA, in base pairs. Length (BP) 9162 8144 7126 6108 5090 4072 3054 Migration Distance (cm) 5.56 7.84 10.12 13.50 17.93 23.79 32.06 15.27
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4. An oligodeoxynucleotide 10 bases in length was subjected to the Maxam Gilbert procedure for sequence determination. The decamer was initially labeled with 32P (as phosphate) at the 50 -end. The four individual sequencing reactions were carried out, and the mixtures were applied individually to four lanes of a 20%T polyacrylamide gel. Following electrophoresis, the following autoradiographic pattern (Fig. 10.16) was obtained:
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(a) Identify the anodic and cathodic ends of the gel as either the top or bottom of the diagram. At which end were the samples applied (b) Derive the base sequence of the oligodeoxynucleotide, starting from the 50 -end.
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