The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.
We constructed the broad-host-range plasmid pUCD800 containing the sacB gene of BaciUus subtilis for use in the positive selection and isolation of insertion sequence (IS)Y'elements in gram-negative bacteria. Cells containing pUCD80 do not grow on medium containing 5% sucrose tinless the sacB gene is inactivated. By using pUCD800, we isolated a 1.4-kilobase putative IS element from Agrobacterium tumefaciens NTlRE by selection for growth on sucrose medium. This putative IS element'appears to be unique to Agrobacterium strains.
By screening an Arabidopsis expression library with an antiserum against chloroplast envelope proteins, we have isolated a partial cDNA with an open reading frame that encodes a polypeptide similar to P-type cationtransporting ATPases. The corresponding genomic done was isolated and the complete coding sequence was deduced after identification and mapping of introns. Ca2+-ATPases from animals are among the best studied P-type ATPases. Their function is to establish steep Ca2+ gradients across cellular membranes and maintain cytoplasmic Ca2+ at submicromolar concentrations. This allows transient increases in cytoplasmic Ca2+ to mediate the transduction of various signals. These enzymes fall into two categories: Ca2+-ATPases located in the plasma membrane (PM Ca2+-ATPases) and Ca2+-ATPases located in the sarcoplasmic/endoplasmic reticulum membranes (SER Ca2+-ATPases). Primary structure similarity between these two types of enzyme is -30%, whereas similarity within these types is >50%o (8).We have cloned a P-type ATPase unique in both its subcellular localization and its structure. It is found in the plant plastid envelope, a membrane not previously known to contain a P-type ATPase. Its deduced primary structure most closely resembles that of mammalian PM Ca2+-ATPases. It is, however, clearly distinct from the mammalian polypeptides and appears to represent an unusual type of calcium transporter. MATERIALS AND METHODScDNA Cloning. An Arabidopsis thaliana (L. cv. Columbia) shoot cDNA library in AYES (9) was screened with an antiserum against spinach chloroplast envelope proteins in the 55-to 75-kDa size range, and positive clones were isolated and characterized as described (10). This resulted in the cloning and identification of PEAJa (see Fig. 1). Subsequent isolation ofa longer cDNA, PEA]b (see Fig. 1
An ATP-dependent Ca2+ uptake activity was identified in plasma membrane vesicles prepared from Synechococcus sp. strain PCC 7942. This activity was insensitive to agents which collapse pH gradients and membrane potentials but sensitive to vanadate, indicating that the activity is catalyzed by a P-type Ca(2+)-ATPase. A gene was cloned from Synechococcus sp. strain PCC 7942 by using a degenerate oligonucleotide based on a sequence conserved among P-type ATPases. This gene (pacL) encodes a product similar in structure to eukaryotic Ca(2+)-ATPases. We have shown that pacL encodes a Ca(2+)-ATPase by demonstrating that a strain in which pacL is disrupted has no Ca(2+)-ATPase activity associated with its plasma membrane. In addition, Ca(2+)-ATPase activity was restored to the delta pacL strain by introducing pacL into a second site in the Synechococcus sp. strain PCC 7942 chromosome.
INTRODUCTIONThe equilibration step serves to saturate the IPG strip with the SDS buffer system required for the second-dimension separation. The equilibration solution consists of buffer, urea, glycerol, reductant, SDS, and dye. The buffer (50 mM Tris-HCl, pH 8.8) maintains the appropriate pH range for electrophoresis. Urea and glycerol are added to reduce the effects of electroendosmosis, thus helping improve protein transfer from the IPG strip to the second dimension. The reductant (dithiothreitol) ensures that disulfide bridges are broken. SDS ensures that the proteins are denatured and also provides a net negative charge to all proteins. Iodoacetamide, introduced during a second equilibration step, alkylates thiol groups on the proteins, preventing their reoxidation during electrophoresis, and thus reducing streaking and other artifacts in the second-dimension separation. Iodoacetamide also alkylates residual dithiothreitol, preventing point streaking and other silver staining artifacts. Finally, a tracing dye (bromophenol blue) is added to allow the electrophoresis to be monitored during the run.
Biological samples may contain contaminants that interfere with analysis by two-dimensional (2-D) electrophoresis. Lysates or biological fluids are complex mixtures that contain a wide variety of nonprotein substances in addition to the proteins to be analyzed. These substances often interfere with the resolution of the electrophoretic separation or the visualization of the result. Macromolecules (e.g., polysaccharides and DNA) can interfere with electrophoretic separation by clogging gel pores. Small ionic molecules can impair isoelectric focusing (IEF) separation by rendering the sample too conductive. Other substances (e.g., phenolics and lipids) can bind to proteins, influencing their electrophoretic properties or solubility. In many cases, measures to remove interfering substances can result in significantly clearer 2-D patterns with more visible spots and better resolution. It should be borne in mind, however, that analysis of samples by 2-D electrophoresis is usually most successful and informative when performed with minimally processed samples, so it is important that any steps taken to remove interfering substance be appropriate to the sample and only performed when necessary. Procedures for the removal of interfering substances therefore represent a compromise between removing nonprotein contaminants, and minimizing interference with the integrity and relative abundances of the sample proteins. This chapter presents a number of illustrative examples of optimized sample preparation methods in which specific interfering substances are removed by a variety of different strategies.
The vacuolar H+-ATPase of higher plants is a member of the V-ATPase family, which comprises complex, multisubunit ATPases found in a11 eukaryotes. The electrochemical gradient created by the V-ATPase is thought to provide the driving force for the iecondary transport of other ions and metabolites (Taiz, 1992). In barley (Hordeum vulgare L.) roots this enzyme may be involved in the sequestration of Na+ and Ca2+ ions in the vacuole, because the proton gradient produced by the ATPase is used by Na+/H+ and Ca2+/H+ antiports to drive the uptake of Na+ and of Ca2+ (Garbarino and DuPont, 1989; DuPont et ai., 1990). The quatemary structure of the ATPase from barley roots is very similar to that from other organisms, with approximately 10 different subunits (DuPont and Momssey, 1992).Two of the best-characterized subunits of the V-ATPases are the A and B subunits of the large, knob-like head group on the cytoplasmic side of the vacuolar membrane. The A subunit is between 67 and 70 kD, and the B subunit is between 53 and 60 kD in various organisms. Both A and B subunits are present in a stoichiometry of three per enzyme complex and contain ATP binding sites, although the B subunit is not directly involved in ATP hydrolysis (Puopolo et al., 1992). The primary structures of the A and B subunits are highly conserved among widely divergent organisms, making them useful for evolutionary studies. Isoforms of the B subunit have been described for mammals, and the distribution of the isofonns seems to be tissue specific (Puopolo et al., 1992). It was of some interest to leam whether there are also isoforms of the B subunit in plants.Two different cDNA clones for the B subunit of the barley V-ATPase, designated HTBl and HTB2, were selected from a barley root cDNA library. The two clones were very similar to each other, having higher sequence identity to each other than to any other B subunit sequence in the GenBank data base (see Table I for details). Since barley is an inbred diploid organism, it is likely that HTBl and HTB2 are encoded by different genes, rather than alleles of the same gene. The next most similar sequence was that for the Arubidopsis
INTRODUCTIONFollowing first-dimension IEF and equilibration of the IPG gel strips, the proteins are separated on the basis of their molecular weight in the second dimension on an SDS-PAGE gel. Systems for this separation are available from a variety of suppliers and are commonly found in many protein chemistry laboratories. This protocol describes a method for placement of the IPG strip and gives some recommended electrophoresis conditions for these second-dimension gels.
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