High resolution two-dimensional electrophoresis of basic as well as acidic proteins.

A previously described two-dimensional electrophoresis procedure (O'Farrell, 1975) combined isoelectric focusing and sodium dodecylsulfate slab gel electrophoresis to give high resolution of proteins with isoelectric points in the range of pH 4–7. This paper describes an alternate procedure for the first dimension which, unlike isoelectric focusing, resolves basic as well as acidic proteins. This method, referred to as nonequilibrium pH gradient electrophoresis (NEPHGE), involves a short time of electrophoresis toward the cathode and separates most proteins according to their isoelectric points. Ampholines of different pH ranges are used to optimize separation of proteins with different isoelectric points. The method is applied to the resolution of basic proteins with pH 7–10 Ampholines, and to the resolution of total cellular proteins with pH 3.5–10 Ampholines. Histones and ribosomal proteins can be readily resolved even though most have isoelectric points beyond the maximum pH attained in these gels. The separation obtained by NEPHGE with pH 3.5–10 Ampholines was compared to that obtained when isoelectric focusing was used in the first dimension. The protein spot size and resolution are similar (each method resolving more than 1000 proteins), but there is less resolution of acidic proteins in this NEPHGE gel due to compression of the pattern. On the other hand, NEPHGE gels extend the range of analysis to include the 15–30% of the proteins which are excluded from isoelectric focusing gels. The distribution of cell proteins according to isoelectric point and molecular weight for a procaryote (E. coli) was compared to that of a eucaryote (African green monkey kidney); the eucaryotic cell proteins are, on the average, larger and more basic.     

Application of zwitterionic detergents to the solubilization of integral membrane proteins for two-dimensional gel electrophoresis and mass spectrometry

Comparative analysis has long been utilized in biological research to interpret protein interactions in both drug na?ve versus drug challenged and normal versus diseased tissues. The technology of proteomics today allows researchers to provide insight into old and still open questions related to biological mechanisms while offering the opportunity to discover novel details in cellular lifecycles. Perhaps the most powerful way to execute these differential displays is in the combination of two-dimensional (2-D) gel electrophoresis and mass spectrometry. While these two techniques together are well suited for abundant and soluble proteins found in cells, rare proteins and integral membrane proteins are still problematic. Recently, a series of novel zwitterionic detergents has been reported in the literature that shows a substantial improvement in solubilizing integral membrane proteins. We show that the amidosulfobetaine, 4-octylbenzol amidosulfobetaine, is better than 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS) at solubilizing both an ion channel and a G-protein coupled receptor (GPCR), while another amidosulfobetaine, myristic amidosulfobetaine (ASB-14), was better than CHAPS at solubilizing a GPCR. Neither membrane protein was visible after staining with colloidal Coomassie blue, silver nor Sypro Ruby. However, a comparison against a duplicate immunoblot allowed for the localization and identification of the ion channel from a 2-D gel by liquid chromatography-tandem mass spectrometry.     

Efficient extraction of proteins from woody plant samples for two-dimensional electrophoresis

Protein extraction from plant samples is usually challenging due to the low protein content and high level of contaminants. Therefore, the 2-DE pattern resolution is strongly influenced by the procedure of sample preparation. Efficient solubilization of proteins strictly depends on the chaotrope and detergent in the extraction buffer. Despite the large number of detergents that have been developed for the use in protein extraction and IEF, there is no single compound able to efficiently extract proteins from any source. Hence, optimization has to be performed for each type of sample. We tested several chaotrope/detergent combinations to achieve optimal solubilization and separation of proteins from Norway spruce [Picea abies (L.) H. Karst.] needles and European beech (Fagus sylvatica L.) leaves and roots. The same chaotrope mixture (7 M urea, 2 M thiourea) was found to be suitable for the extraction and separation of proteins from all samples. Nonetheless, the efficiency of the surfactants tested varied between samples so that optimal extraction and separation was achieved with different detergents or combination of detergents for each sample. The 2-DE separation of spruce needle proteins was optimal in a mixture of two zwitterionic detergents (2% CHAPS and 2% decyl dimethylammonio propanesulfonate). Beech proteins were best separated in buffers containing sugar-based detergents (2% n-octyl beta-D-glucopiranoside in the case of leaf samples and 2% dodecyl maltoside for the root samples). IEF was performed in buffers with the same composition as the extraction buffer except for the root proteins that were better focused in a buffer containing 2% CHAPS.     

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.     

Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry

A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.     

Polyacrylamide gel electrophoresis and immunoblotting of proteins extracted from paraffin-embedded tissue sections

We show in this communication that polyacrylamide gel electrophoresis (PAGE) and immunoblotting of proteins can be performed using one to two 5-7 micron paraffin sections of tissues fixed in non-cross-linking fixatives (acetone, alcohol, or modified Carnoy's solution). Proteins for study were extracted from paraffin sections of mouse foot pad and liver. The presence of unaltered keratin polypeptides in tissues fixed with either acetone or alcohol was demonstrated in gels stained with Coomassie brilliant blue. The preservation of their antigenic determinants was demonstrated with immunoblotting. Furthermore, the immunoreactivity of soluble proteins, such as albumin, remained unaltered in immunoblots obtained from paraffin-embedded mouse liver sections. These data indicate that tissues embedded and stored in paraffin are useful for the above-mentioned biochemical and immunological studies and may therefore be an important technique for diagnostic pathology or retrospective studies.     

Triple-spot proteins in two-dimensional gel electrophoresis.

A triple-spot pattern of polypeptides occurring in two-dimensional gel electrophoresis of proteins is described. The presence of a mutant protein, Pc 1 Duarte, which results in a splitting of all three polypeptides, is evidence that they are produced by the same gene. This pattern is seen in about 1% of the proteins from a variety of sources. Typically, about 50% of the protein occurs as a single major spot, the remainder occurring as two polypeptides with an additional negative charge and slightly different molecular weight. The reproducibility of this pattern implies a functional significance which is presently unknown. The implication of this configuration for patterns seen by one-dimensional gel electrophoresis is discussed.     

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.     

Separation of histones from contaminating ribosomal proteins by two-dimensional gel electrophoresis

Sea urchin histones can be separated from ribosomal proteins by two-dimensional gel electrophoresis. Electrophoresis on Triton X-100/6 m urea gels in the first dimension results in preferential retardation of the histones, which then migrate more rapidly than ribosomal contaminants on SDS gel electrophoresis in the second dimension. The advantages and generality of the system are discussed.     

Preparation of membrane proteins for analysis by two-dimensional gel electrophoresis

In order to separate hydrophobic membrane proteins, we have developed a novel two-dimensional electrophoresis system. For the iso-electric focusing, agarose was used as a supporting matrix and n-dodecyl-beta-D-maltopyranoside was used as a surfactant. In combination with a previously developed Tris/MES electrophoresis system in the second dimension, distinct spots were reproducibly detected from hydrophobic membrane proteins whose grand average hydropathicity (GRAVY) exceed 0.3. In contrast to the immobilized pH gradient system, c-type heme was also visualized in this system.     

pK-matched running buffers for gel electrophoresis.

Electrophoresis through agarose and polyacrylamide-type gels is the standard method to separate, identify, and purify nucleic acids. Properties of electrophoresis buffers such as pH, ionic strength, and composition affect performance. The buffers in use contain a weak acid or weak base buffered by a compound with a dissimilar pK. Herein, three pK-matched buffers were developed, each containing two effective buffering components: one weak base and one weak acid which have similar pKa at 25 degrees C (within 0.3 pK units): (i) Ethanolamine/Capso, pH 9.6; (ii) triethanolamine/Tricine, pH 7.9; and (iii) Bis-Tris/Aces, pH 6.7. On agarose gels, the buffers in various concentrations were tested for separation of double-stranded DNA fragments with various DNA markers, agarose gel concentrations, and field strengths. Mobility was inversely proportional to the logarithm of molecular weight. The buffers provided high resolution without smearing at more dilute concentration than is possible with standard TAE (Tris/acetate, pH 8.0) or TBE (Tris/borate, pH 8.3) buffers. The buffers were also tested in 7 M urea denaturing LongRanger sequencing gels and in nondenaturing polyacrylamide SSCP gels. The pK-matched buffers provide good separation and high resolution, at a broad range of potential pH values. In comparison to TAE and TBE, pK-matched buffers provide higher voltage and current stability, lower working concentration, more concentrated stock solutions (up to 200x), and lower current per unit voltage, resulting in less heat generation.     

Method for extraction of proteins from green plant tissues for two-dimensional polyacrylamide gel electrophoresis

A method has been developed for the extraction of proteins from green plant tissues to be used in two-dimensional polyacrylamide gel electrophoresis. Three purification steps were necessary to overcome the problems due to streaking, charge heterogeneity, and other artifacts: After it was ground in liquid nitrogen, the powdered material was stirred in the presence of insoluble polyvinylpyrrolidone for binding phenols, and sodium ascorbate for binding quinones; proteins were precipitated with ammonium sulfate, and the sample was dialyzed. Hundreds of proteins could be detected after Coomassie blue staining using 200 micrograms of proteins from apical buds of Sinapis alba L.     

Study on proteins from yeast cytoplasmic ribosomes by two-dimensional gel electrophoresis

Summary Proteins of yeast cytoplasmic ribosomes were analyzed by two different methods of two-dimensional gel electrophoresis: run at pH 8.6 in 1-D¹ and at pH 4.6 in 2-D (Method A); run at pH 5.0 in 1-D and in the presence of sodium dodecyl sulfate in 2-D (Method B). The numbers of proteins estimated were 28 (Method A) and 29 or 30 (Method B) in the 40S small subunit, and 40 (Method A) and 41 (Method B) in the 60S large subunit, respectively. Molecular weights of proteins in the small and the large subunits were found to be less than 40,000 and 60,000 respectively.     

Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis

A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P-40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a greater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when membrane proteins were extracted with phenol and precipitated with ammonium acetate in methanol; background staining and streaking were diminished and proteins were clearly resolved. This method makes possible the resolution required for meaningful qualitative and quantitative comparisons of protein patterns on two-dimensional gels of plant membrane proteins.     

Very-high-resolution two-dimensional gel electrophoresis of proteins using giant gels

An improved high-resolution two-dimensional gel system for separating complex protein mixtures is described that allows a threefold increase in the number of proteins detected. Like the original O'Farrell system, proteins are separated in the first dimension by isoelectric point and in the second dimension by size. The improved resolution results primarily from a 2.5-fold increase in the size of both dimensions. Although best resolution is obtained by application of <100 μg of protein containing >5 × 10⁶ cpm, as much as 150 μg of protein may be applied without appreciable loss of resolution. Useful separations may be made with up to 1.5 mg. By doubling the thickness of both dimensions, as much as 3 mg of protein can be separated into 300–400 separate peaks.     

An optimized protocol for isolation of soluble proteins from microalgae for two-dimensional gel electrophoresis analysis

Two-dimensional gel electrophoresis (2-DE)is a core proteomic technique to studyprotein expression and function in livingorganisms. Although it has been extensivelyused for investigation of bacterial, yeast,animal and plant tissue cells, there islimited information about the use of 2-DEin microalgal research. In this study, anumber of key chemical reagents, includingacetone, trichloroacetic acid, urea,thiourea, dithiothreitol, and tributylphosphine, were quantitatively evaluatedfor 2-DE of green microalgae, using Haematococcus pluvialis as a model system.The goal was to maximize the number andstaining intensity of protein spots whileminimizing streaking and smearing on thesecond dimensional SDS gel. Compared tonon-frozen immobilized pH gradients (IPG)strips, freezing of the IPG strips at –20 ^°C after isoelectric focusing (IEF)enhanced protein resolubilization andtransfer into the SDS gel, and thusimproved resolution while eliminatingvertical point streaking on the SDS gel. Itwas also confirmed that manipulation ofsample loading capacity is a simple,effective purification strategy forselective investigation of the proteins ofinterest and of varying abundances. Theprotocol was also successfully applied toprofiling protein expression in H.pluvialis under external stressconditions, indicating its potentialusefulness in further proteomics studies ofthis organism and related species.     

The use of ASB-14 in combination with CHAPS is the best for solubilization of human brain proteins for two-dimensional gel electrophoresis

Daniel Martins-de-Souza, Bruno Menezes de Oliveira,