Multiphasic Buffer System Research Articles

SDS disc electrophoresis of proteins in homogeneous, low‐concentrated polyacrylamide gels

In the attempt to separate in a single gel run low- and high-molecular-weight proteins, we present here a multiphasic buffer system designed for this purpose. It avoids the continuous stacking of SDS as it occurs in the 'classical' SDS-PAGE. The system allows complete stacking and destacking of proteins in the 3.5-250 kDa range at acrylamide concentrations as low as 4.5% T (total acrylamide concentration in %) and 2.6% C (degree of cross-linking in %). Taurine is used as the trailing ion in the cathode buffer and in the resolving zone of the gel, and two different counterions (Tris and imidazole) in the stacking zone. The gel system is easy to prepare and, due to the very low acrylamide concentrations, it is ideal for analytical as well as for preparative tasks.

A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteins.

A new, versatile, multiphasic buffer system for high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins in the relative molecular weight range of 300 000-3000 Da is described. The system, based on the theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins in the above M(r) range. The buffer system uses taurine and chloride as trailing and leading ion, respectively, and Tris, at a pH close to its pK(a), as the buffering counterion. Coupled with limited variation in the acrylamide concentration, this electrophoresis system allows to tailor the resolution in the 6-200 kDa M(r) range, with minimal difficulties in the post electrophoretic identification processes.

Electrophoretic Separation of βA4 Peptides (1–40) and (1–42)

Different sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) systems designed for the separation of peptides were compared for their usefulness in separating synthetic β-amyloid peptides βA4 (1–40) and βA4 (1–42). Clear resolution was achieved by addition of 8Murea to the separation gel and use of a multiphasic buffer system employing bicine and sulfate as trailing and leading ions, respectively (bicine/Tris/urea gels). Under these conditions, the longer peptide migrated faster than the one ending at amino acid 40. The usefulness of this SDS–PAGE system for the analysis of βA4-related peptides generated during cellular metabolism was demonstrated by immunoprecipitation and electrophoretic separation of radiolabeled peptides secreted by cells transfected with amyloid precursor protein cDNAs.

Rapid analysis of mitotic histone H1 phosphorylation by cationic disc electrophoresis at neutral pH in minigels

A new method is described for analysis of histone H1 and other basic proteins by cationic disc electrophoresis in polyacrylamide gels at neutral pH. The multiphasic buffer (disc) system uses Na + as leading ion, l-histidine as trailing ion, and Hepes as buffering counterion. These “Hepes/histidine gels” have three advantages over conventional acid-urea gels for studies of H1 phosphorylation and dephosphorylation: speed, convenience, and the need for only small amounts of cells or chromatin. Core histones and their acetylated forms can also be separated in gels containing 0.4% Triton X-100. The difference in electrophoretic mobility between mitotic (superphosphorylated) and interphase H1 from HeLa cells is approximately twice as great at neutral pH as at pH 4.5, making it possible to separate these two H1 forms rapidly and easily in Hepes/histidine “minigels” only 5-cm long. Total histones can be rapidly prepared by simply neutralizing 0.2 n HCl extracts, and the entire analysis, from harvesting cells to destaining gels, can be carried out in 1 day. The stacking effect of the disc system produces sharp bands and high resolution even with relatively dilute samples.

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.

Electrophoretic purification of soluble trehalase from Drosophila melanogaster by selective unstacking

Specific activity of soluble trehalase from Drosophila melanogaster has been increased at least 10 times by preparative electrophoresis using selective unstacking. However, enzyme yield was poor, with only 17% of the original activity recovered. A comparison of K m of crude and partially purified trehalase revealed no difference (0.650 and 0.666 m m, respectively). Electrophoretic parameters for a modified version of multiphasic buffer system 4014 are described and electrophoretic parameters which may be utilized in the electrophoretic purification of trehalase from 17 species of Drosophila are reported.

Continuous optical scanning in polyacrylamide gel electrophoresis: Estimation of the apparent diffusion coefficient of β-lactoglobulin B

The continuous scanning apparatus developed by Catsimpoolas was applied to an analysis of the concentration profiles of a protein, β-lactoglobulin B, while it was subjected to polyacrylamide gel electrophoresis (PAGE) in a multiphasic buffer system. Continuous optical scanning in PAGE permitted reliable estimation of the standard deviation of the concentration profile (σ), the relationship between σ 2 and time, and the apparent diffusion coefficient, D′, derived from σ 2, as the current density varied from 2 to 9 mA/cm 2, protein load varied from 250 to 900 μg/cm 2, and the ionic strength varied from 0.015 to 0.065 m. Under these conditions, D′ was linearly related to current density and protein load. Further, log ( D′) was linearly related to gel concentration (% T) ranging from 6 to 14%. However, D′ was nonlinearly related to ionic strength. Due primarily to the ionic strength factor, the apparent diffusion coefficient of protein in gels appeared to be approximately 10-fold larger than under the conditions of high ionic strength conventionally used in sedimentation and diffusion studies. Extrapolation of D′ to 0% T, zero protein load, zero current density, and “infinite” ionic strength (assuming noninteraction of these factors), as well as correction for viscosity and temperature, yielded an estimated free-diffusion coefficient, D 20,w, of 3.1 × 10 −7 cm 2/s, which is compatible with previously reported values. These studies indicate that the optimal resolution obtained by PAGE will be considerably lower than that predicted theoretically on the basis of free-diffusion coefficients, and suggest that electrostatic interaction between the proteins and/or deformation of voltage gradient and pH within the protein zones may contribute significantly to band spreading.

Amino acid spacing in isotachophoresis on polyacrylamide gel: A critical evaluation

Isotachophoresis of colored model proteins was carried out on polyacrylamide gel, using the stack of a multiphasic buffer system computed on the basis of the Jovin theory and operative at pH 10.4. The stack was elongated by milligram loads of various amino acids per analytical gel. The extent of the stack was determined by chemical localization of the leading and trailing constituents. The isotachophoretic nature of the stack was ascertained by determining the positions of all protein specles under study as intermediate between the leading and trailing constituents. The shallow pH gradient across the extended stack was measured. Spacing of proteins in isotachophoresis is restricted, with regard to constituent multiplicity and constituent load, by the practical limitations of electrophoresis time and gel length. Therefore, spacing by a small number of constituents at relatively low loads was attempted. Amino acids were chosen as spacers because they can be selected conveniently, in radioactively labeled form, for the specific separation between pairs of proteins with particular mobilities. In the particular buffer system used, lysine, histidine, serine, and threonine were found to be effective spacers between bovine serum albumin (BSA) and hemoglobins, while no amino acids effectively spaced between hemoglobins A and S. It is concluded that specific spacing in isotachophoresis on polyacrylamide gel (ITPPA) can be useful in improving resolution. However, in practice, few spacers exist in the mobility range of proteins, the positions of which relative to specific proteins could be convenlently located on the basis of isotope analysis or other assays. Mixtures of multiple spacers with undefined mobilities appear applicable as “blind spacers” only at concentrations so low that their effectiveness is annulled.

Polyacrylamide gel electrophoresis with optical scanning, using multiphasic buffer systems: The stack

The zone of stacked protein in polyacrylamide gel electrophoresis (PAGE) generated by a multiphasic (discontinuous) buffer system was analyzed by continuous optical scanning during electrophoresis. In agreement with theoretical prediction, zone width was found to be constant with time; band area was linearly related to protein load; band dispersion, measured as the standard deviation (σ) of the concentration profile, was independent of current density between 1 and 8 mA/cm 2; and σ decreased with increasing ionic strength of the gel buffer. The protein concentration within the stack ranged from 13 to 34 mg/ml for β-lactoglobulin B of 36,000 mw. In lieu of the predicted uniform distribution (“square-wave” function) for peak shape, the observed values of kurtosis and skewness indicated a slightly flattened Gaussian profile of the protein concentration in the stack.

Studies of cholesterol binding in the soluble fraction of the adrenal cortex of the guinea pig

A cholesterol-binding component has been studied in the supernatant of guinea pig adrenocortical cytosol after heating for two minutes in a boiling water bath. Enzymatic degradation revealed that the binding component was a protein. On sucrose density gradient analysis the cholesterol-binding protein had an S value of approximately 2, and a similar binding component was not present in unheated cytosol. Cholesterol binding to heated adrenal cytosol was unaffected by sulfhydryl reactants. The equilibrium association constant at 0°C was approximately 10 4 M −1 and the concentration of binding sites was 300 × 10 −9 mol per mg protein. Heated adrenal cytosol was found to bind not only cholesterol but also cholesterol sulfate and 20α -hydroxycholesterol when examined by sucrose density gradient analysis. When examined by polyacrylamide gel electrophoresis, it appeared that cholesterol and cholesterol sulfate were bound to the same protein band. On the other hand binding of 20α-hydroxycholesterol was not detected with the multiphasic buffer system that was used. Heated adrenal cytosol did not bind cholesterol oleate, pregnenolone, and pregnenolone sulfate; nor did it bind cyclic AMP, phosphatidyl choline, NADP, acetyl coenzyme A, or prostaglandin E 2. Cholesterol binding in heated cytosol was not confined to the adrenal; similar binding was found in liver, kidney, lung, testis, muscle, gut, and brain.

A Catalogue of Isohormones of Human Growth Hormone based on Quantitative Polyacrylamide Gel Electrophoresis

With the growing recognition that all polypeptide hormones exist in heterogeneous forms, i.e., isohormones, this study represents the initial development of a comprehensive catalogue of the various isohormone species of human growth hormone (hGH) in relation to molecular structure. The catalogue is based primarily on results obtained in quantitative polyacrylamide gel electrophoresis (PAGE) in a multiphasic buffer system optimized for characterization of hGH. Thus, hGH forms can be recognized in objective, numerical terms based on physicochemical parameters of molecular size and net charge. Isoelectric focusing in polyacrylamide gel; and PAGE in sodium dodecyl sulphate in the presence and absence of reducing agents supplement the quantitative PAGE criteria. Specific hGH immunoreactivity is necessary to completely distinguish hGH from hormones of similar size and charge properties (prolactin and chorionic somatomammotrophin). In the examination of eight standard hGH preparations obtained from four laboratories, all preparations exhibited multiple isohormone species, from two to five components per preparation. These identifiable components appear to represent the predominant species of a bidirectional continuum of hGH components, the continuum being in regard to both molecular size and charge. Within any given preparations, the continuum presents itself as a series of identifiable "charge isomers". Between preparations, in addition, there are size differences as well, which may relate to conformational changes (unfolding) in some preparations.

Natural pH gradients formed by amino acids: Ampholyte distribution, time course, use in electrofocusing of protein, relation to pH gradients in isotachophoresis, and separator effects

Natural pH gradients can be formed from as few as seven amino acids when a polyacrylamide gel, containing a mixture of the 0.01 m amino acids, is placed in an electric field between 0.01 m anolyte and catholyte solutions of the isoelectric “leading” and “trailing” amino acids in the mixture. The amino acids seem to distribute at or near the anodic and cathodic pH gradient terminals, as well as in a position along the pH gradient approximating their isoelectric point ( pI). Only the two amino acids at the terminals in contact with identical electrolytes at their pIs are isoelectric and distribute in single peaks. The electrofocusing at or near its pI of a protein in a pH gradient formed by amino acids was demonstrated. Stacking of the seven amino acids (0.015 mmol each) in a multiphasic buffer system operative at pH 10.4 gives rise to a spatially extended pH gradient within the stack suitable for “cascade stacking.” After transposition of the extended stack between isoelectric “leading” (anolyte) and “trailing” (catholyte) constituents, this pH gradient was maintained. Amino acid electrofocusing provides a tool for the comprehensive physical-chemical analysis of pH gradients and electrofocusing by measurement of the positions, peak widths, and peak shapes of defined carrier ampholytes as a function of time.

Experimental Validation of the Predicted Properties of a Multiphasic Buffer System Applied to Polyacrylamide Gel Electrophoresis

Abstract The properties of a buffer system predicted by T. M. Jovin's theory of multiphasic (discontinuous) buffer systems have been tested experimentally both in free solution and in polyacrylamide gels. The most widely applied multiphasic buffer system, viz., the Tris-glycine system of Ornstein and Davis was used for this purpose. The properties of this system were computed on the basis of the Jovin theory. The pH, specific conductance, and boundary displacement were measured in the buffers corresponding to the original and the operative buffers of both the stacking (upper) gel and the separation (lower) gel in polyacrylamide gel electrophoresis. Values of pH and specific conductance in the absence of gels were found to be in reasonable agreement with theory. In polyacrylamide gels, after removal of residual reactants of the polymerization reaction by equilibration with the appropriate buffer, there was good agreement between observed and predicted values of pH and boundary displacement. However, the ob...

Fractionation and characterization of an oligomeric series of bovine keratohyalin by polyacrylamide gel electrophoresis

Keratohyalin has been fractionated and characterized by polyacrylamide gel electrophoresis, in a multiphasic buffer system in the presence of sodium decylsulfate. Keratohyalin behaves as an aggregating series of homologous oligomers, composed of between 1 and 13 (or more) monomeric units. There is a linear relationship between the logarithm of electrophoretic mobility and gel concentration for each member of the homologous series, with a common value for the extrapolated free mobility. The slope of this line, the retardation coefficient, is linearly related to band number and also to molecular weight. The molecular weight of the monomer is estimated to be 20,500 on the basis of the plot of retardation coefficient versus MW. Several methods for estimation of molecular weight by polyacrylamide gel electrophoresis are discussed. The 13-member oligomeric series of keratohyalin was isolated by use of selective stacking.