Separation of t-RNAs by two-dimensional polyacrylamide gel electrophoresis

A pH 5.8 polyacrylamide gel electrophoresis buffer is described. Electrophoresis in this MES-citrate system at pH 5.8 separates E. coli transfer RNAs into 15 bands using 15% acrylamide gels. Polyacrylamide gel electrophoresis in a second dimension at pH 8.3 further resolves E. coli t-RNAs into 20 spots.     

Identification of initiation factors and ribosome-associated phosphoproteins by two-dimensional polyacrylamide gel electrophoresis.

A two-dimensional polyacrylamide gel electrophoresis procedure has been used to identify initiation factors rapidly in the high-salt-wash fraction from reticulocyte ribosomes. Initiation factors are identified by relative mobility and by co-electrophoresis with purified factors. A creatine phosphate/ATP/GTP/Pi exchange system is described which has been used to maintain [gamma-32P]ATP and [gamma-32P]GTP at constant specific activity in the cell-free protein-synthesizing system. Phosphorylated proteins associated with the protein-synthesizing complex have been identified using a combination of the two procedures. The salt-wash fraction contains eight major phosphorylated proteins and a number of minor ones. Two phosphorylated proteins are observed to comigrate with two of the three subunits of eukaryotic initiation factor 2 (eIF-2), the initiation factor involved in binding Met-tRNAf onto the 40-S subunit and promoting dissociation of 80-S ribosomes. eIF-4B, one of the proteins involved in binding mRNA to 40-S subunits is also phosphorylated. The remainder of phosphorylated proteins in the high-salt-wash fraction are not previously characterized initiation factors and have not been identified further. Two of the six phosphoproteins associated with the salt-washed ribosomes comigrate with ribosomal proteins; one is the major phosphorylated protein in 40-S ribosomal subunits, the other is an acidic protein.     

Identification of mitochondrial protein complexes inArabidopsis using two-dimensional blue-native polyacrylamide gel electrophoresis

Mitochondria fulfill a wide range of functions in the plant cell, including producing ATP, providing carbon skeletons for biosynthesis, and biosynthesizing vitamins and cofactors. Recently, mitochondria have been implicated in the pathway of programmed cell death in plant cells. In addition, mutations in the mitochondrial genome have been shown to be causally related to cytoplasmic male sterility—the failure to produce functional pollen in a range of crop plants. Proteomics has been used to attempt to catalogue mitochondrial proteins and extend our understanding of this essential organelle. Conventional proteomics based on isoelectric focusing and SDS-PAGE is unsuitable for hydrophobic proteins and therefore does not allow the identification of many components of the respiratory complexes. To identify such proteins, we have used blue-native PAGE to fractionate protein complexes in their native state, followed by SDS-PAGE to separate component subunits of each complex. A total of 40 protein spots were reproducibly resolved, and 29 were identified by means of mass spectrometry, thus giving a map of the most abundant complexes in plant mitochondria. Chaperones; transporters; novel proteins; and proteins involved in the respiratory chain, the citric acid cycle, amino acid and carbon metabolist, and stress response were identified. This study gives new insight on the role and functioning of well-characterised and recently identified mitochondrial proteins by localising them to specific complexes. It also identifies novel proteins in plant mitochondria.     

Micro-scale two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins

Summary A specially designed apparatus and conditions are described for the rapid analysis of ribosomal proteins by two-dimensional gel electrophoresis on a micro scale. The resolution of proteins in electropherograms is comparable to that obtained with other systems, but because of miniaturization, only 0.5 to 1 g of each protein is required, and the entire procedure, including electrophoresis in both dimensions, and staining and destaining can be completed in 6 to 7 hours.     

Comparison of plant cytoplasmic ribosomal proteins by two-dimensional polyacrylamide gel electrophoresis

The electrophoretic mobilities of the cytoplasmic ribosomal proteins of several species of plants were compared using two-dimensional electrophoresis. The total number of proteins as well as the number of acidic and basic proteins in individual species varied markedly. Of the species examined, Triticum aestivum had the highest number of basic cytoplasmic ribosomal proteins and Hordeum vulgare had less than half as many. However, marked similarities were noted in the electrophoretic mobilities of many of the proteins, especially for wheat, rye, and barley and for peas and beans. There was a statistically significant positive correlation between the numbers of basic proteins in the species and their chromosome number.     

Preparative two-dimensional polyacrylamide gel electrophoresis of 32 P-labeled RNA

Complex mixtures of RNA molecules may be separated by two-dimensional electrophoresis on polyacrylamide gel slabs. The first dimension of the separation is carried out on acid gels in the presence of a high urea concentration, the second on more concentrated gels buffered at pH 8. The method has been applied to the complete separation of RNA fractions obtained after a preliminary gel electrophoresis of partial enzymic digests of ³²P-labeled bacteriophage RNA. Another application is the fractionation of partial digests as obtained in sequence determination of RNA molecules. Spots are detected by autoradiography and extracted by a simple micro procedure which yields the material in a concentrated form suitable for sequence analysis by fingerprinting.     

Modification of commerical and custom-built slab gel electrophoresis units for two-dimensional polyacrylamide gel electrophoresis

Many commercial and custom-built slab gel electrophoresis units can be modified to function as two-dimensional polyacrylamide gel electrophoresis units with the insertion of Plexiglas adapters. These adapters can be made for about $50 a pair and can be used for either temporary or permanent modification of the slab gel units. The physical dimensions of the adapters can be varied to permit great flexibility in the diameter of cylinder gels and the thickness of slab gels that can be run together. For example, proteins from 6-mm cylinder gels can be easily separated on 1-mm slab gels, which can then be dried for autoradiography.     

Identification and mapping of human saphenous vein medial smooth muscle proteins by two-dimensional polyacrylamide gel electrophoresis

Changing smooth muscle phenotype and abnormal cell proliferation are important features of vascular pathology, including the failure of saphenous vein bypass grafts. We have characterised and mapped protein expression in human saphenous vein medial smooth muscle, using two-dimensional (2-D) polyacrylamide gel electrophoresis. The 2-D system comprised a nonlinear immobilised pH 3-10 gradient in the first dimension (separating proteins with isoelectric point values between pH 3-10), and 12%T total gel concentration sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension (separating proteins in the range 14,000-200,000 Daltons). Using a combination of peptide mass fingerprinting by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and partial amino acid sequencing by nanospray tandem mass spectrometry, a subset of 149 protein spots was analysed, with 129 protein spots being identified and mapped. The data presented here are an important addition to the limited knowledge of venous medial smooth muscle protein expression in vivo. Our protein map will facilitate the identification of proteins differentially expressed in human saphenous vein bypass grafts. In turn, this may lead to the elucidation of molecular events involved in saphenous vein bypass graft failure. The map should also provide a basis for comparative studies of protein expression in vascular smooth muscle of varying origins.     

Double-beam flying spot scanner for two-dimensional polyacrylamide gel electrophoresis

The construction of a double-beam photometer in which the light source is a cathode ray oscilloscope is described. The light spot from the oscilloscope was focused and reduced in size at the gel plane to give a diameter of less than 0.15 mm and make it possible to scan over a 50 X 59-mm rectangle; using reduced spatial resolution (spot less than 0.2 mm) the area scanned becomes 70 X 90 mm. The light from the CRT was divided into two beams; one was directed through the transparent object to a photomultiplier and the other to a reference photomultiplier. The signals from these two detectors were converted to the logarithm of the ratio by a logging amplifier to give a direct measure of absorbance. Positioning of the spot, control of light intensity, and measurement of absorbance were carried out through an interface to a 16-bit computer. The relationship between measured and actual absorbance was linear over the range of absorbance 0 to 2, which could be raised to 1 to 3 by placing a neutral filter in the reference beam. The system generated an image containing 256 X 256 pixels in about 5 min, the scanning speed was determined by the persistence time of the P4 phosphor on the cathode ray tube, and faster scans can be made using A6 phosphor.     

Probing hairpin structures of small DNAs by nondenaturing polyacrylamide gel electrophoresis

The influence of temperature on the electrophoretic mobility of small DNAs, capable of forming hairpin structures, is investigated under nondenaturing conditions. Three series of hairpin-forming DNAs containing different numbers of thymidine, deoxyadenosine, and deoxyguanosine residues in their loop, and an identical sequence in the helical region, are analyzed. All show enhanced electrophoretic mobility if they adopt the hairpin conformation. The same quantitative relationship between hairpin formation and increase in electrophoretic mobility is observed for all of the three series. The constancy of this increase suggests a dependence of electrophoretic acceleration on the length of the helical region. A possible application of nondenaturing electrophoresis is monitoring the hairpin/coil transition. Another possible application is the detection of dimers formed by partially self-complementary sequences. This dimer formation is detected for completely complementary DNAs, whereas sequences which might form imperfect double helices, especially those with three bulged-out nucleotides, prefer hairpin formation. The possible applications are experimentally approached and discussed.     

Resolution of rat mitochondrial matrix proteins by two-dimensional polyacrylamide gel electrophoresis.

The mitochondrial matrix subfractions from rat liver, kidney cortex, brain, heart, and skeletal muscle were isolated and their protein components were resolved by two-dimensional polyacrylamide gel electrophoresis, revealing between 120 and 150 components for each matrix subfraction. Excellent resolution was obtained utilizing a pH 5 to 8 gradient in the first dimension and in 8 to 13% exponential acrylamide gradient in the second dimension, increasing the number of mitochondrial matrix proteins observed 3-fold over one-dimensional systems. Protein components tentatively identified by co-migration with pure enzymes and by known tissue distributions are carbamoyl-phosphate synthetase (EC 2.7.2.5), ornithine transcarbamylase (EC 2.1.3.3), glutamate dehydrogenase (EC 1.4.1.3), pyruvate carboxylase (EC 6.4.1.1), citrate synthase (EC 4.1.3.7), fumarase (EC 4.2.1.2), aconitase (EC 4.2.1.3), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2), dihydrolipoyl transsuccinylase (EC 2.3.1.12), lipoamide dehydrogenase (EC 1.6.4.3), glutamate-aspartate aminotransferase (EC 2.6.1.1), and the two subunits of pyruvate dehydrogenase (EC 1.2.4.1). Protein components unambiguously identified by peptide mapping are citrate synthase, aconitase, and pyruvate carboxylase. The inner membrane subfraction from rat liver mitochondria was also resolved two dimensionally; the alpha and beta subunits of ATPase (F1) (EC 3.6.1.3) were identified by peptide mapping.     

Subpicogram analysis of sweat proteins using two-dimensional polyacrylamide gel electrophoresis

Sweat collected from six normal volunteers was analyzed to determine if reproducible protein patterns could be obtained using two-dimensional polyacrylamide gel electrophoresis of ¹²⁵I-labeled sweat proteins. This method has the capability of easily detecting picogram quantities of protein. Once the methods of collection of the sweat had been standardized, reproducible patterns were obtained from these volunteers. Over 100 discrete spots were revealed by a combination of fluorography and rare earth screen radioautography of dried two-dimensional gels. This method will allow analysis of sweat for qualitative and quantitative variations in protein content in pathologic conditions such as cystic fibrosis, renal failure, and diabetes.     

Aldosterone-induced proteins in toad urinary bladders. Identification and characterization using two-dimensional polyacrylamide gel electrophoresis

Aldosterone-stimulated Na+ transport is mediated by new protein synthesis, but the identification of specific aldosterone-induced proteins (AIPs) has proven difficult and the cellular function of such proteins is unknown. Using high resolution two-dimensional polyacrylamide gel electrophoresis and autoradiography we have identified AIPs of similar isoelectric points (5.8 to 6.4) and molecular weights (70,000 to 80,000) in membrane-rich and cytosolic subcellular fractions of epithelial cells derived from single toad urinary bladders. The ability of actinomycin D to inhibit both AIP synthesis and aldosterone-induced Na+ transport is consistent with a role for these proteins in the natriferic action of aldosterone. In addition, since non-natriferic concentrations of cortisol did not induce similar proteins, AIP synthesis appears to be mineralocorticoid-specific. The relationship of AIP synthesis to Na+ transport was also studied. Since amiloride, which blocks Na+ transport in high resistance epithelia, did not affect the synthesis of these proteins, Na+ transport is not required for their synthesis. In addition, similar proteins were not induced when Na+ transport was stimulated by antidiuretic hormone and theophylline. Consequently, AIP synthesis is not merely a nonspecific consequence of the cellular metabolic changes associated with Na+ transport.