Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis

One of the most useful techniques for visualization of the proteome is two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-D SDS-PAGE). This technique possesses unmatched resolving power for separation of proteins [2-4] and has been used extensively to analyze proteins [5-8], their regulation [9-18], and posttranslational modifications [19-22], Several tech-... 

Fig. 3. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [ S]methionine polypeptides from cholic acid-induced (A) and control (B) cultures of Eubacterium V.P.I. 12708. Arrows indicate the synthesis of new polypeptides.

Ramnath, M., Beukes, M., Tamura, K., and Hastings, J.W. (2000). Absence of a putative mannose-specific phosphotransferase system enzyme IIAB component in a leucocin A-resistant strain of Listeria monocytogenes, as shown by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Appl Environ Microbiol 66, 3098-3101. 

A fully automated two-dimensional electrophoresis (2DE) system for rapid and reproducible protein analysis is described. 2DE that is a combination of isoelectric focusing (lEE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein expression analysis. Here, all the operations are achieved in a shorter time and all the transferring procedures are performed automatically. The system completed the entire process within 1.5 h. A device configuration, operational procedure, and data analysis are described using this system. 

Two-dimensional electrophoresis (2DE) that is a combination of isoelectric focusing (lEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (4) is widely used for protein expression analysis. 

The combination of CIEF and mass spectrometry is analogous to two-dimensional electrophoresis [4]. In this case, the mass spectrometer provides the molecular-weight information instead of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This information can be obtained by on-line CIEF-MS [5] or by using CIEF as a micropreparative technique [6]. 

Nestler, H.P. and Doseff, A. (1997) A two-dimensional, diagonal sodium dodecyl sulfate polyacrylamide gel electrophoresis technique to screen for protease substrates in protein mixtures. Anal. Biochem. 251, 122-125. 

Fibroblasts from these patients express the normal number of surface LDL receptors which exhibit normal binding capacity. Moreover, immunodetection of LDL receptors analyzed by two-dimensional isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that internalization-defective receptors are indistinguishable from those of normal cells [105]. Despite the presence of the normal number of intact receptors, fibroblasts from these patients are unable to take up LDL by receptor-mediated endocytosis. The mutation thus appears to be in some aspect of the receptor internalization mechanism [87]. Clinical symptoms resemble those of receptor-negative or receptor-defective FH. 

Note The elution should be optimized by checking at least two to three different elution solvents. Eluates may be assessed using a one-dimensional (ID) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel stained with colloidal Coomassie. 

A combination of circular dichroism, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chemical crosslinking, and analytical ultracentrifugation studies showed that both the apo- and metallated derivatives of H21(31-mer) form two-stranded a-helical coiled coils in aqueous solution. Further characterization of these derivatives by EPR spin-label experiments helped to determine its three-dimensional backbone structure. In these studies, a Cys-21 mutant of the 31-mer coiled coil, H21/C21(31-mer), was prepared and labeled with a thiol-specific nltroxide spin label (MTSL = l-oxyl-2,2,5,5-tetramethyl-A -pyrroline-3-methyl-methanethiosulfonate) at position 21 of the peptide sequence which is the site of metal substitution in the ET heterodimer. Comparison of the low-temperature, dipolar-broadened spectrum of the spin-labeled dimer with those of magnetically dilute peptide samples yielded a backbone-to-backbone distance that was nearly identical to that of the GCN4 homodimer. Based on these results, computer modeling studies provided an estimate of the metal-to-metal distance in the ET heterodimer of m-m > 25 A. The electron-transfo properties of this system are now being studied by a combination of laser flash-quench and pulse radiolysis techniques. 

Separation of the target protein(s) using one- or two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) or HPLC techniques. 

This chapter will describe in detail the procedure for Western blotting of polypeptides and proteins separated on a denaturing polyacrylamide gel system. This procedure is used routinely in our laboratory for the analysis of polypeptides from a variety of subcellular fractions of whole tissue and cell lines, and has evolved over a number of years in the hands of several people. Many different immunoblotting procedures are currendy available details of variadons from the I-protein-A method described here are given in the Notes secdon, as are brief amendments covering electrotransfer from two-dimensional and isoelectric focusing systems. A detailed descripdon of sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) is not appropriate for this chapter, and the reader is referred to vol. 1, Chapter 6, and refs. 9-14. For details of the producdon of polyclonal and monoclonal andsera, Chapters 1-6 in this vol. 

Macfarlane, D.E. (1989) Two dimensional benzyldimethyl-n-hexadecylaimnonium chloride-sodium dodecyl sulfate preparative polyacrylamide gel electrophoresis a high capacity high resolution technique for the purification of proteins from complex mixtures. Anal. Biochem. 176,457-463. 

T8. Tuszynski, G. P., Buck, C. A., and Warren, L., A two-dimensional polyacrylamide gel electrophoresis (PAGE) system using sodium dodecyl sulfate—PAGE in the first dimension. Anal. Biochem. 93, 329-338 (1979). 

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