One-dimensional gel electrophoresis

Camden, Australia An improved one-dimensional gel electrophoresis method was used to separate three waxy proteins in wheat starch 261... 

The arrangement used for two-dimensional gel electrophoresis may comprise various combinations of apparatus used for one-dimensional gel electrophoresis already described. Cylindrical gels used for the first dimension may be conveniently adapted to Akroyd type gels ( 8.4.2.1) (Martini and Gould 1971). Alternatively, strips of Akroyd... 

Figure 8 Canine serum. One-dimensional gel electrophoresis of dog serum separated on SDS-PAGE followed by silver staining. Track N is from a normai heaithy dog and track M is from a dog with a monocionai gammopathy (igA) Prominent H and L chains can be cieariy seen. (Courtesy of Miiier i, University of Veterinary Medicine, Vienna.)...

FIGURE 3 One-dimensional gel electrophoresis (Laemmli s, 15% acrylamide) of histone... 

Prokaryotic cells express hundreds to thousands of proteins while higher eukaryotes express thousands to tens of thousands of proteins at any given time. If these proteins are to be individually identified and characterized, they must be efficiently fractionated. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has typically been use to study protein mixtures of <100 proteins. Onedimensional electrophoresis is useful because nearly all proteins are soluble in SDS, molecules ranging from approximately 10,000 to 300,000 molecular weight can be resolved, and extremely basic or acidic proteins can be visualized. The major disadvantage to one-dimensional gels is that they are not suitable for complex mixtures such as proteins from whole cell lysates. 

Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P. (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203, 173-9. 

Additional reagents and equipment for one- or two-dimensional gel electrophoresis (e.g., unitb3.i) and for staining membranes (unitb3.3)... 

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-stain-ing, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method. 

Fig. 4. Two-dimensional gel electrophoresis. The protein sample is first subjected to isoelectric focusing in one dimension and then to SDS-PAGE in the second dimension.

Protein samples are separated by one-dimensional SDS-PAGE or two-dimensional gel electrophoresis in polyacrylamide gels. The separated proteins are then transferred (blotted) to a nitrocellulose or nylon sheet. This is incubated with specific antibody to the protein and then unbound antibody is washed away. Those proteins in the gel that bind the antibody are detected either by autoradiography (if the specific antibody was radiolabeled) or by using a second labeled antibody that binds to the primary antibody. 

Two-dimensional gel electrophoresis is the method of choice for analysing the products of crosslinking experiments. The usual one-dimensional electrophoretic techniques are rarely useful except in the simplest cases, Huang and Richards (1977), for example, used slab gels to follow the... 

The stimulation of steroid synthesis in gonadal cells is dependent on ongoing protein synthesis. Experiments with protein synthesis inhibitors have shown the presence of one or more proteins with half lives of 5-15 min which are essential for the expression of the stimulating hormone effect on steroidogenesis [42,45-47]. Many attempts to identify the protein(s) have been made but none have been detected that satisfy all the functional and kinetic criteria required. Using two-dimensional gel electrophoresis a 28 kDa protein has been identified recently [48], however its functional properties are unknown. It is also not known if the active protein is synthesised de novo or is derived from a precursor protein by covalent modification [49]. 

Two-dimensional gel electrophoresis One-dimensional electrophoresis Multidimensional liquid chromatography Various types of chromatographic fractionation Affinity probe isolations... 

Proteins are separated by one-dimensional, or more often, two-dimensional gel electrophoresis. The separated proteins are subjected to in situ tryptic digestion, and the peptides are separated by liquid chromatography and identified by mass spectrometry. See Nishihara, J.C. and Champion, K.M., Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain, Electrophoresis 23, 2203-2215, 2002. 

Galeva, N. and Altermann, M. (2002) Comparison of one-dimensional and two-dimensional gel electrophoresis as a separation tool for proteomic analysis of rat liver microsomes cytochromes P450 and other membrane proteins. Proteomics 2, 713-722. 

Low, TY, Seow, TK. and Chung, M.C. (2002) Separation of human erythrocyte membrane associated proteins with one-dimensional and two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Proteomics 2,... 

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