One- and Two-Dimensional Gel Electrophoresis

Our next step was to establish the general applicability of the microfluidic device for real biological samples. Proteins obtained from ID and 2D gel separation of Saccharomyces cerevisiae were used to evaluate the device. 

We next analyzed proteins obtained from a 2D gel separation of a total yeast lysate. Four peptides were identified from HXKA yeast along with one peptide from keratin and four peptides from trypsin. All the peptides identified had an XCOII higher than 2.0 except for the ion MH+ at mfz 630.4 which was a small peptide observed as a 1+ ion. The amount of protein present in the gel was estimated to be 200-300 fmol. 

The Coupling of Microfabricated Fluidic Devices with Electrospray Ionization 

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One- and two-dimensional gel electrophoresis (ID- or 2D-GE) is an important tool in the separation and isolation of intact proteins [9], In ID-GE, the proteins are separated in a sodium dodecylsulfate poly(acrylamide) gel (SDS-PAGE). The separation is according to molecular weight. In 2D-GE, the proteins are first separated by isoelectric point (pi, isoelectric focussing, lEF), and next by molecular weight. 2D-GE is considered to be the most powerful tool in protein separation. Nevertheless, the technique suffers from problems it is labour-intensive, analysis time is long, and the reproducibility poor. Furthermore, hydrophobic proteins do not behave well in the first lEF step and tend to form broad bands. 

The TM isoform content of a variety of smooth muscle tissues has been examined by one- and two-dimensional gel electrophoresis, including aorta, pulmonary artery, carotid, trachealis, esophagus, duodenum, jejenum, taenia coli, rectum, and others (Fatigati and Murphy, 1984 Yamaguchi et al., 1984 Xie et al., 1991). In all cases, two major components were observed to be present in close to equal amounts. The designation of these as a and P was made on the basis... 

Lupin proteins have been analyzed by a number of different techniques, including liquid chromatography (Duranti et al., 1995), differential sedimentation (Franco et al., 1997 Freitas et al., 2000), proteomic analysis by one- and two-dimensional gel electrophoresis (Duranti et al., 1992 Magni et al., 2005b) or liquid chromatogra-phy/electrospray ionization tandem mass spectrometry (Schwend et al., 2003 Wait et al., 2005 Magni et al., 2007), isoelectrofocusing (Quaresma et al., 2007), crystalb-zation (Biesiadka et al., 1999), and expression of recombinant protein (Scarafoni et al., 2001). 

Cells or subcellular fractions were harvested in SDS and urea containing lysis buffer exactly as described [5], and total cellular homogenates analyzed by one- and two-dimensional gel electrophoresis [7]. The gels were both stained with Coomassie brilliant blue-R and processed for fluorography as described [5]. 

As one- and two-dimensional gel electrophoresis are often classified as planar chromatographic techniques and are increasingly more exploited in proteomic and molecular biology studies, and for medical diagnostic purposes as well, the book also covers electrophoretic-MS methods and applications. 

Proteomics data derived from one- and two-dimensional gel electrophoresis and mass spectrometry [81] combined with the genome sequence of the plant pathogen Xylella fastidiosa [82] revealed a putative protein, which showed some... 

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

Rosenfeld, J., Capdevielle, J., Guillemot, J.C. and Ferrara, P. (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Analytical Biochemistry 203, 173-179. 

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. 

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,... 

Analysis typically involves three steps. First, exoproteins are isolated from bacterial cultures. The aim of this process is to retain as many proteins as possible while eliminating components that inhibit isoelectric focusing. To do so, proteins are enzymatically treated and precipitated to remove salts and other non-protein components. Second, the proteins are resolved by one- or two-dimensional gel electrophoresis (see Fig. 1). Third, proteins are visualized and the composition and quantities of protein spots determined. The protocol described below uses a mini-gel format that is relatively fast and requires small culture volumes compared with large-format two-dimensional gel electrophoresis. 

R. Aebersold, J. Leavitt, R. A. Saavedra, L. E. Hood, and S. B. H. Kent, Internal amino acid seqnence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in sitn protease digestion on nitrocellnlose, Proc. Natl. Acad. Sci. USA 84, 6970-6974 (1987). 

In Situ Digestion and Desorption from Membranes. While the electrospray ionization technique enjoys a considerable advantage as an online chromatographic (HPLC) detector, the direct desorption of proteins and peptides from electroblotted membranes using MALDI provides an equally important link to one-dimensional and two-dimensional gel electrophoresis. In such a scheme the laser beam would... 

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. 

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]. 

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