Structural analysis of recombinant proteins prepared by semi-dry electroblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Proteins that were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were electroblotted onto polyvinylidene difluoride membranes in procedures to prepare homogeneous recombinant proteins for direct N-terminal sequence analysis. A semi-dry blotting procedure was employed to immobilize protein bands on the membranes for subsequent sequence analysis. This method has been used routinely to evaluate the quality of recombinant proteins, which are present in crude cell extracts produced by different expression systems or under different expression conditions. N-Terminal processing, amino acid misincorporation, as well as the inefficient secretion of recombinant proteins can be detected by direct N-terminal sequence analysis of the purified electroblotted samples. Consequently, time-consuming chromatographic procedures can be eliminated. These procedures are also especially valuable for determining degradation sites of a purified recombinant protein, as well as evaluating multiple gene products expressed by isolated cluster genes.     

Barley cultivar discrimination: I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and glycoprotein blotting.

Two different methods of detecting electroblotted glycoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Tris-buffer soluble barley seed proteins were examined for their applicability for barley cultivar discrimination. These are the highly specific, lectin-based concanavalin A/peroxidase method and the more general periodate/danyslhydrazine method. The results of the periodate/dansylhydrazine method enabled us to divide the 20 examined cultivars into three groups, whereas the more sensitive concanavalin A/peroxidase method revealed six different glycoprotein patterns. In comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the alcohol-soluble barley seed proteins (hordeins) gave nine different banding patterns. A combination of hordein electrophoresis together with glycoprotein staining by the concanavalin A/peroxidase method made it possible to classify the cultivars into twelve groups, the largest of which contained four cultivars. The qualitative expression of the glycoprotein patterns seemed to be independent of growth conditions, whereas the band intensities obviously were not. As a whole, glycoprotein blotting is a valuable supplement to sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hordeins in barley cultivar discrimination.     

Efficient extraction of proteins from recalcitrant plant tissue for subsequent analysis by two‐dimensional gel electrophoresis

Protein extraction for two‐dimensional electrophoresis from tissues of recalcitrant species is quite problematic and challenging due to the low protein content and high abundance of contaminants. Proteomics in Shorea robusta is scarcely conducted due to the lack of a suitable protein preparation procedure. To establish an effective protein extraction protocol suitable for two‐dimensional electrophoresis in Shorea robusta, four procedures (borate buffer/trichloroacetic acid extraction, organic solvent/trichloroacetic acid precipitation, sucrose/Tris/phenol, and organic solvent/phenol/sodium dodecyl sulfate) were evaluated. Following these, proteins were isolated from mature leaves and were analyzed for proteomics, and also for potential contaminants, widely reported to hinder proteomics. The borate buffer/trichloroacetic acid extraction had the lowest protein yield and did not result in any banding even in one‐dimensional electrophoresis. In contrast, organic solvent/phenol/sodium dodecyl sulfate extraction allowed the highest protein yield. Moreover, during proteomics, organic solvent/phenol/sodium dodecyl sulfate extracted protein resolved the maximum number (144) of spots. Further, when proteins were evaluated for contaminants, significant (77–95%) reductions in the nucleic acids, phenol, and sugars were discernible with refinement in extraction procedure. Accumulated data suggested that the organic solvent/phenol/sodium dodecyl sulfate extraction was the most effective protocol for protein isolation for proteomics of Shorea robusta and can be used for plants that have a similar set of contaminants.     

Separation of durum wheat proteins by ultrathin-layer isoelectric focusing: a new tool for the characterization and quantification of low molecular weight glutenins

An isoelectric focusing method capable of resolving all groups of storage protein of the wheat seed, including the most basic low molecular weight glutenin (LMWG), was developed. Ultrathin polyacrylamide gels were used after drying and rehydration with 8 M urea, 50 mM DTE and 2.4% carrier ampholytes (pH 4-9). Densitometric scanning of the isoelectric focusing gels permitted a more accurate and specific quantitation of LMWG components among various cultivars than patterns based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two main genetic types (i.e. 'gamma-42' and 'gamma-45') of durum wheats were separated on the basis of the proportion in LMWG in storage proteins, but no significant difference was found within these groups. Advantages of the system as regards reliability, high resolution, ability to abolish protein oxidation and preventing reaggregation of LMWG were also discussed.     

An efficient method for purification of nonspecific lipid transfer protein‐1 from rice seeds using kiwifruit actinidin proteolysis and ion exchange chromatography

Plant nonspecific lipid transfer proteins are small basic proteins that transport phospholipids between membranes and are subdivided into two subfamilies, nsLTP₁ (9 kDa) and nsLTP₂ (7 kDa). LTPs have potential application in the defense reactions against pathogens and the drug delivery systems. Many efforts have been made for purification of different nsLTPs from various plants; however, most of them used successive purification procedures. We have developed a relatively simple and efficient method for the purification of rice nsLTP₁, based on the proteolytic activity of kiwifruit actinidin on the rice seed extract and one‐step chromatographic procedure on a CM‐Sepharose column. The purity of protein was determined by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). The isolated LTP₁ migrated as a homogenous polypeptide with molecular mass of 9 kDa that confirms the efficiency of actinidin on the digestion of major contaminations present in the rice seed extract without any harmful effect on the LTP₁. The advantages of using proteolytic activity of actinidin in purifying rice LTP₁ includes the reduced separation time allowing the purification of LTP₁ in one‐step chromatographic procedure, low costing, high efficiency, and the relative simplicity of the method.     

A new multiphasic buffer system for sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100,000-1000, and their detection with picomolar sensitivity.

A novel multiphasic buffer system for high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dansylated and nondansylated proteins/peptides in the relative molecular mass (Mr) range of 100,000-1000 is described. The system, based on Jovin's theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins/peptides within the above Mr range. The buffer system uses Bicine and sulfate as trailing and leading ion, respectively, and Bistris and Tris as counter ions in the stacking and separating phase, respectively. Through selection of two different counter ions--the characteristic feature of the present ionic system--the stacking limits of a multiphasic buffer system can be further widened, thus making it applicable to gel electrophoresis of a larger spectrum of rapidly migrating species, such as sodium dodecyl sulfate-proteins/peptides and nucleic acids, than has been possible previously. Highly sensitive detection methods for proteins as well as for polypeptides down to approximately Mr 1000 are described. Dansylated proteins/peptides were detected by their fluorescence either directly within the gel or following electroblotting into anion-exchange or polyvinylidene difluoride membranes. The latter procedure resulted in detection sensitivities of approximately 1 ng. Nondansylated proteins/peptides were either detected within the gel by colloidal Coomassie staining or by electroblotting into polyvinylidene difluoride membranes, followed by colloidal gold staining. Prior to both staining procedures the proteins/peptides were pretreated with glutardialdehyde in the presence of borate at near neutral pH values to generate protein/peptide polymers of poor solubility. For a given pH the efficiency of the latter procedure was significantly influenced by the nature of the buffer ion used in the fixation buffer. In contrast to conventional fixation procedures even small polypeptides (Mr 1000) were immobilized and approximately 15 ng and 0.75 ng could be detected after colloidal Coomassie and colloidal gold staining, respectively.     

Free solution capillary electrophoresis of proteins using untreated fused-silica capillaries.

Numerous efforts have been made to separate proteins by capillary zone electrophoresis (CZE). The most common optimization techniques are changing the pH of the running buffer, coating the capillary surface with a hydrophilic polymer, or using additives in the sample solution. Surface coatings and solution additives can reduce the adsorption of the protein onto the capillary surface, but they diminish the separation efficiency and the resolution of CZE. This paper reports the successful separation of proteins in a untreated fused-silica capillary by raising the pH of the running buffer and washing between runs with 1.0 M sodium hydroxide. Under these conditions, model proteins and proteins in human serum have been determined by CZE. It is shown that the results from CZE are compatible with those of sodium dodecyl sulphate-polyacrylamide gel electrophoresis.     

Analysis of expression and comparative profile of normal placental tissue proteins and those in preeclampsia patients using proteomic approaches

Preeclampsia (PE) is a complex and serious condition of pregnancy. Trophoblasts in human placenta can be separated and collected by laser capture micro-dissection (LCM). Protein in trophoblasts have been extracted and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), finally 962 unique proteins are identified by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Comparison of differential expressed proteins in normal and those in PE are investigated. Two-dimensional electrophoresis (2DE) and MS were used to identify differential expressed proteins. 13 differential expressed proteins include signal transduction protein, molecular chaperone, cell skeleton proteins are identified, in which 3 proteins are down-regulated and 10 proteins are up-regulated. They might be correlated with the cause of PE.     

Study of grasshopper meiosis-associated proteins using two-dimensional polyacrylamide gel electrophoresis

Premeiotic and meiotic whole testes from grasshoppers were compared for the presence of meiosis associated proteins using one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. One-dimensional sodium dodecyl sulphate-polyacrylamide gels detected differences between premeiotic and meiotic samples but two-dimensional gels gave more precise results. Isoelectric focusing revealed only one meiosis-associated protein, while nonequilibrium pH gradient electrophoresis detected five more. It is not known whether these proteins relate to the nuclear aspects of meiosis, or associated cellular changes. These proteins have been electrophoretically purified and monoclonal antibodies are being prepared.     

Analysis of proteins stained by Alexa dyes

Alexa dye staining of proteins is used for the fluorescence microscopy of single particles that are sometimes multimolecular protein complexes. To characterize the staining, post-staining determination must be made of which protein(s) in a complex have been Alexa-stained. The present communication describes the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for performing this determination. The Alexa-stained proteins are observed directly in gels by illumination with an ultraviolet transilluminator. The test multimolecular particle is bacteriophage T7. The protein capsid of T7 is a multimolecular complex that has both external and internal proteins. SDS-PAGE of Alexa-stained bacteriophage T7 produces fluorescent capsid proteins each of which usually comigrates with an unstained protein. However, one Alexa-induced modification of protein migration was observed by SDS-PAGE. Mass spectrometry shows that the protein with modified migration is the major protein of the outer shell of the T7 capsid. The procedures used are generally applicable. The distribution of Alexa staining among T7 capsid proteins depends on the size of the dye molecule used. The larger the dye molecule is, the greater the preference for external proteins.     

The use of two-dimensional gel electrophoresis in the analysis of organ-specific maize proteins

Two-dimensional gel electrophoresis with non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide gels in the second dimension has been used for the analysis of organ-specific proteins in maize. The method has been used to study the whole protein pattern of developing organs and adult leaves as well as protein patterns of in vitro translation. Examples of two-dimensional immunoblotting and in vitro translation of endosperm-specific proteins are also shown. Two-dimensional gel electrophoresis appears as an essential analytical step in the identification of organ-specific proteins and for the detection of the protein products related to organ-specific genes identified by other means.     

Electrophoresis and chromatography of wheat proteins: available methods, and procedures for statistical evaluation of the data.

Analysis of gluten proteins from the wheat grain endosperm has long challenged the analytical chemist. Several hundred unique polypeptides are present, many in large polymers. This complexity, plus useful relationships of composition to genotype and quality, encouraged development and application of electrophoresis and chromatography for gluten analysis. We review the methods of polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and high-performance liquid chromatography available for study of wheat proteins. Singly and in combination, they provide rapid, reproducible, high-resolution separations based on size, charge, or surface hydrophobicity. As challenging and important as the analyses themselves, however, is interpretation of data. Subjective evaluation is sometimes possible, but statistical methods such as similarity scores, clustering, principal components, multiple linear regression, and partial least squares now are increasingly used for data analysis. We review the use of these procedures, and precautions necessary to avoid misinterpretation of data. Optimal evaluation of protein analytical data will enhance the value of such analyses in wheat breeding, marketing, and processing.     

Separation of proteins from stressed rice (Oryza sativa L.) leaf tissues by two-dimensional polyacrylamide gel electrophoresis: induction of pathogenesis-related and cellular protectant proteins by jasmonic acid, UV irradiation and copper chloride

We have used three kinds of stresses, including the signaling compound jasmonic acid, an environmental stressor, UV irradiation, and a heavy metal salt copper chloride, to study changes in the protein patterns in rice (Oryza sativa L.) leaf tissues using two-dimensional polyacrylamide gel electrophoresis. However, instead of using lysis buffer containing urea (O'Farrell, J. Biol. Chem. 1975, 250, 4007-4021) for extraction of proteins from rice seedling tissues, we used Tris-HCl buffer (commonly used for extraction of proteins for separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) for extraction of proteins and resolved these extracted proteins by the usual method of O'Farrell. Furthermore, the induction of a large number of proteins was clearly observed over controls. No spots corresponding to these induced proteins were found in the control experiment, indicating qualitative changes in protein patterns after various stress treatments. A total of 12 out of 13 proteins could be N-terminally sequenced from jasmonic acid-treated rice leaf tissues, and one protein was sequenced from UV-irradiated leaf tissues. These proteins showed high homology to pathogenesis-related (thaumatin-like protein, a PR5 class protein; a beta-1,3-glucanase precursor; an intracellular PR protein encoded by PBZ1 gene, and an antifungal protein) and cellular protectant (glutathione transferase, EC 2.5.1.18; and ascorbate peroxidase) proteins, from plants, including rice. Results presented here suggest a role for jasmonic acid in the self-defense mechanisms of rice plants.     

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of human parotid salivary proteins.

The proteins in human parotid saliva have been separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis into 20 or more well resolved species. The Coomassie Brilliant Blue (CBB) R-250 and silver staining procedures have been modified to overcome the problems encountered with staining of proline-rich proteins. By means of the CBB R-250 procedure which stains proline-rich proteins pink-violet, immunoblotting, concanavalin A binding, periodate-Schiff staining and zinc binding, all of the major proteins have been characterised. Substantial individual-to-individual differences were observed in the protein patterns formed. Comparison of parotid, submandibular, and whole saliva from a single individual indicated that fewer proline-rich proteins are expressed in submandibular saliva than in parotid, but whole saliva contains much lower levels than either duct secretion. The results will form a useful base for future research into the functions of salivary proteins.     

Sodium dodecyl sulfate versus acid-labile surfactant gel electrophoresis: comparative proteomic studies on rat retina and mouse brain

A long-chain derivative of 1,3-dioxolane sodium propyloxy sulfate, with similar denaturing and electrophoretic properties as SDS, and facilitated protein identification following polyacrylamide gel electrophoresis (PAGE) for Coomassie-stained protein bands, has been tested. Comparative acid-labile surfactant/sodium dodecyl sulfate two-dimensional (ALS/SDS 2-D)-PAGE experiments of lower abundant proteins from the proteomes of regenerating rat retina and mouse brain show that peptide recovery for mass spectrometry (MS) mapping is significantly enhanced using ALS leading to more successful database searches. ALS may influence some procedures in proteomic analysis such as the determination of protein content and methods need to be adjusted to that effect. The promising results of the use of ALS in bioanalytics call for detailed physicochemical investigations of surfactant properties.     

Evaluation of membranes used for electroblotting of proteins for direct automated microsequencing

Protein purification and characterisation have been age-old problems for the biochemist. A new era has arisen with the advent of one- and two-dimensional gel electrophoresis and high sensitivity automated protein microsequencing. These two tools along with electroblotting have made it possible to separate and analyse complex protein mixtures. We studied six different membranes compatible with Edman degradation chemistry to determine their efficiencies at binding proteins electroblotted from one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Their overall blotting-sequencing properties were also evaluated. We found that the polyvinylidene difluoride-based membranes out-performed the glass-based and polypropylene-based membranes under our selected experimental conditions. The problems associated with electroblotting and microsequencing are discussed.     

Extraction of plant proteins for two-dimensional electrophoresis

Three different extraction procedures for two-dimensional electrophoresis of plant proteins are compared: (i) extraction of soluble proteins with a nondenaturing Tris-buffer, (ii) denaturing extraction in presence of sodium dodecyl sulfate at elevated temperature allowing the solubilization of membrane proteins in addition to a recovery of soluble proteins, and (iii) a trichloroacetic acid-acetone procedure allowing the direct precipitation of total proteins.     

Matrix-assisted laser desorption/ionization time-of-flight based wheat gliadin protein peaks are useful molecular markers for wheat genetic study

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation has been used to analyze wheat seed gliadins as an alternative to other established methods, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), etc. The MALDI-TOF approach has shown to have many advantages such as high resolution, cost effectiveness and high throughput. MALDI-TOF-based gliadin profiles have been used for fast wheat cultivar identification. However, the genetic information represented by individual gliadin peaks has not been utilized. In this study a wheat doubled haploid population with a genetic linkage map of good coverage was used to assay individual gliadin peaks from MALDI-TOF profiles as molecular markers. Eight segregating peaks in the population were scored as polymorphic across the population. The 1 to 1 segregating ratios validated the scoring of the peaks and all peaks were mapped to the expected chromosomes or linkage groups on the available linkage map: 1 peak on chromosome 1A, 1 on 6A, 4 on 6B and 2 on 6D.     

Capillary electrophoresis of proteins for proteomic studies

Analyses of proteins in complex mixtures such as cell lyzates are presently performed mainly by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. For structural analysis, each protein in a spot is digested with proteases and the fragment peptides are subjected to Edman sequencing and/or mass spectrometry. These works aim at the total analysis of proteins in a complex mixture and reconstruction of their cooperative functions. Genomic studies are now being combined with these proteomic studies. This review article focuses on the application of capillary electrophoresis aiming at the total analysis of complex protein systems or structural analysis of each separated protein. From this viewpoint, articles on capillary zone electrophoresis, capillary isoelectric focusing, and sieving SDS capillary electrophoresis are reviewed. Since these techniques of capillary electrophoresis have been thoroughly reviewed previously, papers published in 1997 and 1998 are mainly covered.     

Effect of strong detergents and chaotropes on the detection of proteins in two-dimensional gels.

The solubilization of a particular protein is mandatory for its subsequent resolution and detection in two-dimensional gels. However, the extraction solutions, that are compatible with the first-dimensional separation step, such as urea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), do not solubilize all proteins in a sample. We studied the effect of various common, strong detergents and chaotropes, widely used as solubilizing agents, such as sodium dodecyl sulfate, lithium dodecyl sulfate and guanidine hydrochloride, on the solubilization of the total and membrane proteins of the bacterium Haemophilus influenzae. The proteins solubilized with each system were analyzed by two-dimensional electrophoresis and these of interest were identified by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Use of sodium dodecyl sulfate, lithium dodecyl sulfate or guanidine hydrochloride for the solubilization of total proteins of the microorganism resulted in the detection of several additional spots, representing mainly outer membrane proteins, in comparison with those detected in the soluble protein fraction. Solubilization of the proteins of the cell envelope fraction with sodium dodecyl sulfate did not result in a more efficient protein detection when compared to the extraction with the urea/CHAPS system. When the dry immobilized pH gradient strips were rehydrated in a solution containing the proteins of the membrane fraction solubilized with sodium dodecyl sulfate or lithium dodecyl sulfate, a larger number of protein spots were detected in comparison with strips that were rehydrated in the urea/CHAPS solution. However, no improvement was observed in comparison with protein application in sample cups. The additional proteins detected with the use of strong detergents and chaotropes are in the majority difficult to solubilize and less hydrophobic proteins.