Two-dimensional polyacrylamide

Fawcett, JS Sullivan, SV Chrambach, A, Toward a Steady-State Pore Limit Electrophoresis Dimension for Native Proteins in Two-Dimensional Polyacrylamide Gel Electrophoresis, Electrophoresis 10, 182, 1989. 

Mailer, J. L., and Smith, D. S. (1985). Two-dimensional polyacrylamide gel analysis of changes in protein phosphorylation during maturation of Xenopus oocytes. Dev. Biol. 109 150-156. 

Pietrogrande, M.C., Marchetti, N., Tosi, A., Dondi, E, Righetti, P.G. (2005). Decoding two-dimensional polyacrylamide gel electrophoresis complex maps by autocovariance function a simplified approach for proteomics. Electrophoresis 26, 2739-2748. 

O Farrell PH et al. Two-dimensional polyacrylamide gel electrophoretic fractionation. Methods Cell Biol 1977 16 407-420. 

Scharfe C et al. MITOP, the mitochondrial proteome database 2000 update. Nucleic Acids Res 2000 28 155-158. Molloy MP et al. Establishment of the human reflex tear two-dimensional polyacrylamide gel electrophoresis reference map new proteins of potential diagnostic value. Electrophoresis 1997 18 2811-2815. 

Figure 7.1. Effect of GM-CSF on neutrophil protein biosynthesis. Human neutrophils were incubated in RPMI1640 medium supplemented with 2.5% foetal calf serum and 60 jtCi/ml [35S]-methionine, in the presence and absence of 50 U/ml GM-CSF. After 4 h incubation at 37 °C, the cells were pelleted by low-speed centrifugation. The proteins in the cell pellets were precipitated by 10% trichloracetic acid and then analysed by two-dimensional polyacrylamide gel electrophoresis, using iso-electrofocussing in the first dimension. Electrophoresis in the second dimension was performed in the presence of SDS and used a 12% acrylamide gel. Source Experiment of Becky Stringer.

Orphanides G, LeRoy G, Chang CH, Luse DS, Reinberg D (1998) FACT, a factor that facilitates transcript elongation through nucleosomes. Cell 92 105-116 Orrick LR, Olson MO, Busch H (1973) Comparison of nucleolar proteins of normal rat Uver and Novikoff hepatoma ascites cells by two-dimensional polyacrylamide gel electrophoresis. Proc Natl Acad Sci USA 70 1316-1320... 

Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE lEF followed by SDS-PAGE)... 

Craven BA, Totty N, Harnden P, Selby PJ, Banks RE. (2002) Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis evaluation of tissue preparation and sample limitations. Am J Pathol 160, 815-22. 

Nishihara, J.C., Champion, K.M. (2002). Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain. Electrophoresis, 23(14), 2203-2215. 

Dunn, M.J. Corbett, J.M. (1996) Two-dimensional polyacrylamide gel electrophoresis. Methods Enzymol. 271, 177-203. 

Staining of blot transfer membranes permits visualization of proteins and allows the extent of transfer to be monitored. In the protocols described in this unit, proteins are stained after electroblotting from one-dimensional or two-dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes (unitb3.2). PVDF is the preferred, more universal membrane and is emphasized here however, most stains work similarly on nitrocellulose, and many can be used on alternative blotting membranes. 

---