Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins myofibrillar proteins

Electrophoresis of the high-molecular-mass proteins (>500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoresic dye front. Inclusion of 10 mm 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mm 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.     

Improved retention of heme with increased resolution of microsomal proteins in polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis, using lithium dodecyl sulfate instead of the sodium salt, was used to analyze rat hepatic microsomal hemoproteins. Good resolution of hepatic microsomal proteins was obtained with retention of approximately half of the total microsomal heme on the proteins with molecular weights of 45,000 to 50,000. Both the protein resolution and heme retention are better than with electrophoresis procedures previously described. Treatment of rats with chemicals that either increase or decrease microsomal cytochrome P-450 produced proportional changes in the heme associated with the proteins of 45,000 to 50,000 molecular weights on the gel.     

Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins

Electrophoresis of the high-molecular-mass proteins (greater than 500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoretic dye front. Inclusion of 10 mM 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mM 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.     

Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins: improved resolution with Triton X-100

The nonionic detergent Triton X-100 binds in varying proportions to specific ribosomal proteins and decreases the relative mobility of these proteins during electrophoresis. When Triton X-100 binds to these ribosomal proteins in the first-dimension gel, the resolution of the ribosomal proteins in the second-dimension gel pattern is greatly improved. Maximum binding of Triton X-100 to the ribosomal proteins is dependent on pH, urea concentration, and the complete reduction of cysteine and methionine. After first-dimension electrophoresis the Triton X-100 in the gel does not interfere with the binding of sodium dodecyl sulfate to the ribosomal proteins and the molecular weight of these proteins can still be estimated directly from the second-dimension slab gel.     

Assay of plant proteins with bicinchoninic acid for high resolution two-dimensional polyacrylamide gel electrophoresis

A method is described for estimating proteins in the same plant tissue sample that is solubilized for separation by two-dimensional polyacrylamide gel electrophoresis. The method uses a modified bicinchoninic acid (BCA) protein assay procedure and a modified standard urea solubilization buffer to estimate microgram values of unknown protein concentration, in the presence of 9 M urea and 4% Nonidet P-40, from a linear standard curve. A method for a quantitative determination of protein concentration by BCA in a sample containing 9 M urea and 4% Nonidet P-40 is also described. This method is effective for the determination of proteins in minute non-green and green plant tissue, and is especially designed for vegetative and floral shoot apices, and the primordia of inflorescences.     

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the protein, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.     

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.     

Characterization of N-ethylmaleimide-reactive proteins from human tonsillar ribosomes by two-dimensional polyacrylamide gel electrophoresis.

Human tonsillar 80-S ribosomes were 17% and 43% inactivated by 1 mM N-ethylmaleimide after 12 min at 30 or 37 degrees C, respectively. The ribosomes were unaffected by the reagent during the same period of time at 0 or 20 degrees C. 4, 12, 27 and 59 sulfhydryl groups per 80-S ribosomes were found labeled by 1 mM N-ethyl[14C] maleimide after 12 min at 0, 20, 30 or 37 degrees C, respectively. The analysis of radioactively labeled proteins by two-dimensional gel electrophoresis revealed the following: after 3 min at 37 degrees C only two 40-S proteins, S3 and S7, displayed a significant amount of label. After 12 min at 37 degrees C, there was a several-fold increase in the extent of radioactivity found in each of these proteins and, additionally, S1, S2, S4, S5, S15, S22 and S31 were also found among labeled 40-S proteins. S3 appeared to be the most N-ethylmaleimide-reactive 40S protein. After 3 min at 37 degrees C, L10, L17, L20 (and/or S20), L26, L32 and L33, and after 12 min at 37 degrees C, additionally L1, L2, L7, L9, L11, L15, L16, L18, and L25 were labeled among 60-S proteins. l17 and 32 were the most N-ethylmaleimide-reactive proteins under these conditions. After 12 min at 37 degrees C, approx. 26% and 39% of the radioactivity incorporated into the 80 S or 60 S ribosomal protein, respectively, was found in these two proteins. After 12 min at 0 degrees C, S3, L17, L32 and L33 were the only labeled proteins.     

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.     

The rapid electrophoresis of proteins on polyacrylamide gel

A system for express electrophoretic separation of proteins in a thin layer of polyacrylamide gel covalently attached to a glass base has been proposed. A technology of covalent attachment of gel to a glass base has been developed and a small-scale instrument has been constructed which allows to carry out the separation, staining and electrophoregram washing off for 60 min. The quality of the cleaved protein mixtures is not worse than the electrophoregrams obtained using the "Phase System T. M." instrument of "Pharmacia".     

Peptide production from proteins separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis

The development of amino acid sequencers with subnanomolar sensitivities has increased the need for both selective and highly efficient methods for both protein and peptide isolation. In this paper, we describe a simple procedure that utilizes the high resolving capacity of polyacrylamide gel electrophoresis to isolate a single target polypeptide, which can subsequently be subjected to proteolytic digestion and sequencing. Polypeptides are visualized in polyacrylamide gels as dodecyl sulfate/protein complexes, which are passively diffused from gel slices. Free dodecyl sulfate eluted with the protein solution is removed by KCl precipitation, allowing protein digestion with small amounts of trypsin or other proteolytic enzymes. Following enzymatic digestion, the peptide solution is made 6 M guanidine-HCl to remove interfering contaminants and thereby improve resolution of the digest by reverse-phase high-performance liquid chromatography. The peptides generated by this method are suitable for amino acid sequencing with good overall yields, averaging 15-30% on a gas-phase sequenator. The method described is useful for obtaining multiple peptide sequences from a single polypeptide isolated from a complex protein mixture.     

High resolution two-dimensional gel electrophoresis of human erythrocyte membrane proteins.

Three different two-dimensional (2-D) gel electrophoretic techniques have been modified to provide high resolution of human erythrocyte membrane proteins. The resulting gels were referenced to the established one-dimensional (1-D) sodium dodecylsulfate (SDS) gel electrophoretic profile, and the effects of endogenous proteolysis and cytosolic contamination were studied. It is concluded that in vitro proteolysis and cytosolic contamination do not contribute significantly to the patterns observed on the 2-D gels, under the conditions used for erythrocyte ghost preparation. The procedures require only small quantities of blood; as many as twenty 2-D gel profiles can be obtained from 5 ml of blood. The combination of nonequilibrium isoelectric focusing (IEF) in the first dimension, SDS electrophoresis in the second dimension, and very sensitive silver staining techniques resolves more than 250 individual protein spots. This appears to be the most useful single procedure for the analysis of red cell membrane proteins. Membrane protein profiles from patients with Duchenne muscular dystrophy, Wernicke-Korsakoff syndrome, and acanthocytosis with degeneration of the basal ganglia were compared with normal controls. The patterns for Duchenne muscular dystrophy and Wernicke-Korsakoff syndrome were not different from normal patterns. The pattern for the patient with acanthocytosis and degeneration of the basal ganglia consistently showed a high level for one protein in the 100,000 mol. wt. range.     

Visible labeling of proteins for polyacrylamide gel electrophoresis with dabsyl chloride

Several proteins, which are used as molecular weight markers in polyacrylamide gel electrophoresis, were reacted with dabsyl chloride. This labeled them deep orange and the chromophore attachment was stable throughout the electrophoretic procedure and fixation. Small amounts (10-50 micrograms) of the labeled proteins could be loaded onto gels and seen with the unaided eye so that the separation during electrophoresis could be followed. Dabsylation did not affect the mobility of the proteins. The location of the orange band gave a good indication of the position of the protein in the gel so that molecular weight estimations could be made during and immediately following electrophoresis.     

Gradient, polyacrylamide gel electrophoresis of proteins from cytotoxic mycoplasma membranes.

Membranes of $\text{Mycoplasma}\text{pneumoniae}$ were prepared following several different cell lysis procedures (freeze-thaw, french press, digitonin, sonication, osmotic shock, pH shock). All possessed some degree of toxicity for ciliated epithelial cells, but the french press and freeze-thaw processes were optimal in terms of lysis efficiency, membrane yield, and cytotoxicity. Membrane proteins from virulent and attenuated mycoplasmas were electrophoresed with a new, high resolution electrophoresis technique (gradient polyacrylamide gels), and strain differences were detectable among the 50+ discernible bands.     

Direct chemiluminescent imaging detection of human serum proteins in two-dimensional polyacrylamide gel electrophoresis

A novel chemiluminescence (CL)-based imaging method capable of directly detecting proteins in polyacrylamide gels after electrophoresis is proposed. Human serum proteins are presently detected by a direct CL imaging method after native 2-D PAGE. As a consequence, some proteins, including haptoglobin (Hp), Hp precursor, hemopexin (Hpx) precursor, Ig alpha-1 chain C region, and Complement C3 precursor can be detected and identified by MS and MS/MS techniques. These proteins are all acute phase proteins, which have been defined as biomarkers for certain diseases. Moreover, serum proteins from healthy people and cirrhotic patients were analyzed. A decrease in Hp spots for cirrhotic patients could be confirmed. The CL imaging conditions were optimized, including the concentrations of H(2)O(2) and luminol. The process of CL detection of proteins is simple, and there is no need for specialized equipment. In comparison with the traditional CBB-R250 staining method, the detection sensitivity was improved and the detection period decreased about 70 times. Hence, this technique possesses potentials as a rapid, convenient, and inexpensive analytical technique for protein detection and for the diagnosis of diseases.     

Comparison of membrane proteins from benign and malignant human thyroid tissues by two-dimensional polyacrylamide gel electrophoresis

In this study two-dimensional (2D) polyacrylamide gel electrophoresis with silver staining was used to analyze cellular membranous proteins of various normal and pathological human thyroid tissues. The aim was to understand the differences in cellular membranous proteins between these tissues, which would aid in the differential diagnosis of thyroid malignancy. Characteristic protein spots had a molecular mass of 50–64 kDa and a pI of 5.7-6.5. There were two groups of isoform protein spots in this area. The higher-molecular-mass group was found in follicular thyroid cancer tissues which and was not visible in normal thyroid tissues. The low-molecular-mass group was found in follicular carcinoma or adenoma tissues and was detected in one to three spots. The papillary thyroid carcinoma tissues gave different 2D gel maps. There were few spots of papillary thyroid carcinoma tissue membranous proteins within the examined area. The 2D gel maps may be used for differential diagnosis of follicular neoplasm. The characteristics of these protein spots require further investigation.