Molecular weight estimation of proteins by electrophoresis in linear polyacrylamide gradient gels in the absence of denaturing agents.

It was shown that in linear polyacrylamide gradient gels migration distance of a given protein increases as a function of the square root of the time of electrophoresis. The linearity between these two parameters is demonstrated by the statistical analysis of experimental data obtained with proteins of different shapes and a wide range of electrophoretic mobility. The slopes of the regression lines calculated by this method can be utilized to determine the molecular weight of a nondenatured protein. In fact, there is a linear relationship between the log of the molecular weights and the log of the slopes for proteins with M_rs between 20,000 and 950,000.     

Molecular weight estimation using sodium dodecyl sulfate-pore gradient electrophoresis

Pore gradient electrophoresis (PGE) in the presence of sodium dodecyl sulfate (SDS) provides a means for high resolution fractionation of multicomponent protein systems and permits estimation of molecular weights for macromolecules ranging from 10³ to 10⁶. We have evaluated the performance of several methods used to construct calibration curves for estimation of molecular weights using SDS-PGE. A linear relationship between the logarithm of molecular weight, log (M_r), and the logarithm of the relative mobility, log (R_l), can be obtained for a 30-fold range of molecular weights. However, this range of linearity depends on the choice of the concentration gradient, the degree of crosslinking of the gel, and on the nature of the underlying relationship between the retardation coefficient, K_R, and the molecular weight. An empirical relationship, first introduced by Lambin et al. (1976, Anal. Biochem.74, 567) between log (M_r) and the logarithm of the gel concentration at the position reached by the protein, log (%T), provides better linearity over a wider molecular weight range than does the use of log (R_l). We have compared these relatienships by experimental analysis of 10 standard proteins and by a theoretical analysis of an idealized model system. A computer program has been developed which provides appropriate statistical estimation of the molecular weight for an unknown protein, together with its standard error and 95% confidence limits. A new method has also been developed for analysis of nonlinear calibration curves in terms of molecular weight versus distance migrated, based on a theoretically justifiable, physical-chemical model. This model implies that either the relationship between log (M_r) and log (R_l) or the one between log (M_r) and log (%T) will become nonlinear as the range of molecular weight is extended. We suggest that the use of a nonlinear least-squares curve-fitting procedure provides an optimal method for molecular weight estimation when sufficient data are available. Based on these findings, a general strategy is presented for estimation of molecular weights by polyacrylamide gel electrophoresis.     

Reliability of molecular weight determination of proteins by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate

Thirty-four proteins with molecular weights (MWs) between 1.3 × 10⁴ and 9.5 × 10⁵ and seven polypeptide chains of some of these proteins were incubated in sodium dodecyl sulfate and submitted to electrophoresis in a polyacrylamide gradient gel (3 to 30%; relative percentage of bisacrylamide: 8.4%). Analysis of the results confirmed the reliability of the relationship: log(MW) = alog(T) + b, where T is the polyacrylamide concentration reached by a protein, a is the slope, and b is the Y intercept of the regression line. In fact the mean deviation between the expected and the observed MW was ±5.5%. In addition we have shown that MWs of unreduced and reduced proteins can be estimated with the same accuracy and tha MWs of glycoproteins can also be determined with the same accuracy as other proteins.     

Separation of high molecular weight heterodisperse RNA and rRNA by polyacrylamide gels

A polyacrylamide gel containing a low percentage of bisacrylamide is shown to be applicable to separation of RNA molecules within the molecular weight range of 0.7 × 10⁶ to 15 × 10⁶, the molecular weight of giant heterodisperse RNA. The exact relationship between mobility and molecular weight is reported.     

Molecular weights of protein multimers from polyacrylamide gel electrophoresis

A nonlinear relationship between polyacrylamide gel electrophoresis (PAGE) retardation coefficients (K_R) and molecular weights has been observed during analysis of several multimeric proteins. Although the deviation from linearity over a wide range of molecular weights is slight, it can lead to significant errors in the estimation of the sizes of individual multimers. Two alternative methods of analysis of PAGE results are compared and demonstrated to yield linear relationships for multimeric proteins having molecular weights as high as 900,000.     

The use of liquid polyacrylamide in electrophoresis: II. Relationship between gel viscosity and molecular sieving

The effectiveness of molecular size discrimination in liquid polyacrylamide-agar mixed-gel electrophoresis is shown to depend on the polymer chain length. This relationship seems to be mediated by the intrinsic viscosity of the polymer. Possible implications for cross-linked gels are discussed.     

Characterization by polyacrylamide gel electrophoresis of hemoglobins in rabbit embryo, fetus, and adult

The hemoglobin species of rabbit embryo, fetus, and adult were characterized by quantitative polyacrylamide gel electrophoresis (PAGE). Special statistical methods, including joint confidence envelopes for the slope (K_R) and intercept (Y₀) of the Ferguson plot and analysis of covariance were utilized to identify the hemoglobin species. Five embryonic rabbit hemoglobins could be distinguished. Their relative proportions varied with developmental stage. No specific fetal hemoglobin was detected. The two previously known adult hemoglobins were characterized, prepared by isoclectric focusing on polyacrylamide gel (IFPA) and shown to differ by two amino acid substitutions in the β-chain.A general strategy for testing molecular identity by PAGE is outlined.     

Transient state isoelectric focusing. Experimental determination of apparent diffusion coefficients in polyacrylamide gels

A photoelectric scanning assembly utilizing uv absorption optics and an on-line digital data acquisition and processing system has been used to follow kinetically zone spreading during the defocusing stage (absence of electric field) of transient state isoelectric focusing (TRANSIF) in polyacrylamide gels. Measurement of the variance (σ²) of a diffusing zone as a function of time yields a linear relationship, the slope of which corresponds to the apparent diffusion coefficient (D) of the protein. A linear relationship is also obtained when the logarithm of the apparent diffusion coefficients (logD) are plotted vs acrylamide concentration (T). This relationship can be used to extrapolate D to zero gel concentration. The apparent diffusion coefficient measured in this way is significantly larger than the true diffusion coefficient. The slope of the plot logD vs T, designated C_R, is expected to be a measure of molecular size related to the retardation coefficient in polyacrylamide gel electrophoresis.     

Polyethylene Oxide (PEO) and Polyethylene Glycol (PEG) Polymer Sieving Matrix for RNA Capillary Electrophoresis

The selection of sieving polymer for RNA fragments separation by capillary electrophoresis is imperative. We investigated the separation of RNA fragments ranged from 100 to 10,000 nt in polyethylene glycol (PEG) and polyethylene oxide (PEO) solutions with different molecular weight and different concentration. We found that the separation performance of the small RNA fragments (<1000 nt) was improved with the increase of polymer concentration, whereas the separation performance for the large ones (>4000 nt) deteriorated in PEG/PEO solutions when the concentration was above 1.0%/0.6%, respectively. By double logarithmic plot of mobility and RNA fragment size, we revealed three migration regimes for RNA in PEG (300-500k) and PEO (4,000k). Moreover, we calculated the smallest resolvable nucleotide length (N_min) from the resolution length analysis.     

A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels

A sensitive silver stain for detecting bacterial lipopolysaccharides in polyacrylamide gels is developed by modifying the silver-staining method used for proteins (cf. R. C. Switzer III, C. R. Merril, and S. Shifrin, Anal. Biochem.98, 231–237 (1979). Lipopolysaccharides are analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by visualization with either the modified silver stain or periodic acid-Schiff stain. The lipopolysaccharides are stained dark brown by the silver stain. The silver stain is 500 times more sensitive than the periodic acid-Schiff stain and can detect less than 5 ng of rough type lipopolysaccharides. Analyses of 5μg of smooth-type lipopolysaccharides from Salmonella typhimurium and Escherichia coli O111: B4 show each to have 30–40 components of different molecular weights. The use of a lipopolysaccharide having a known structure and variable numbers of repeating units in the O side chain, such as one of the two lipopolysaccharides mentioned above, as molecular weight markers is proposed for the estimation of the molecular weights of other lipopolysaccharides or their components. The lipopolysaccharides can also be stained grayish green, but become grayish blue with a heavy sample load, using a silver-based color-staining method (D. W. Sammons, L. D. Adams, and E. E. Nishizawa, Electrophoresis2, 135–141 (1981)).     

Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.

We describe the use of polyacrylamide gel electrophoresis to estimate chain lengths of double- and single-stranded DNA molecules in the size range 20-1000 base pairs (or nucleotides). Double-stranded DNA molecules of known length produced either by organic synthesis or by restriction endonuclease digestion of viral DNAs were used as standards. The relative electrophoretic mobilities of these standards were examined on both nondenaturing (aqueous) polyacrylamide gels and on denaturing gels containing 7 M urea or 98% formamide. Electrophoretic mobility of DNA is a linear function of the log of molecular weight if appropriate conditions are used, although exceptions are noted. Chain lengths can be conveniently estimated by using as standards bacteriophage gamma DNA restriction fragments or commercially available tracking dyes.     

Identification of macromolecules by quantitative polyacrylamide gel electrophoresis and isoelectric focusing: a comparison of three approaches.

Identification of macromolecules by zone electrophoresis has usually been based on differences in migration distances under a single set of electrophoretic conditions. Classically, it has taken the form of coelectrophoresis on gel slabs. In “quantitative” polyacrylamide gel electrophoresis (PAGE), separation conditions were standardized sufficiently to allow for identification of macromolecules between experiments on the basis of their relative electrophoretic mobilities, R_f ± σ_Rf. More reliably, molecular identity or distinguishability have been based on several R_f values at several gel concentrations (%T) and the linear relationship between log R_f and %T, the Ferguson plot. The slope (retardation coefficient K_R of this plot is desoriptive of molecular size while the γ-intercept (Y₀) is a measure of net charge. The joint 95% confidence envelopes for K_R and Y₀ may be used as criteria for identification of molecules. Distinction between two molecular species depends on the size and position of the two confidence envelopes or ellipses. By pooling estimates of residual varlance (scatter areund the regression line for the Ferguson plot) for several proteins, it is possible to reduce the size of the ellipses and improve the sensitivity of the method to distinguish elesely related species. The sensitivity of this method depends on the size and reprodueibility of the 95% confidence envelopes, and on the limitatiens in the number of electrophoretic fractionations that one is reasonably willing to invest. Any molecular identification problem therefore raises the implieit question whether to base distinction on migration distance, on R_f, or on the joint 95% confidence envelopes for K_R and Y₀ and related statistical (F test) eriteria. Further, in the event of inconsistent answers to the question of molecular distinguishability from the three approaches, we need rational criteria to select the “best” answer. These problems and some solutions are illustrated by the present study which was designed to determine whether the enzymatic digestion products of human growth hormone produced by subtilisin-B are or are not the same as those obtained by digestion with plasmin. It appears that the joint 95% confidence envelopes of K_R and Y₀ provide at this time the most discriminating criteria of distinction, indicating significant differences between nearly all the products of plasmin and subtilisin digestion of hGH. However, the lower resolution provided by the R_f criteria has the advantage that it allows one to group the products of the partial hydrolysis of hGH into “families” which may be associated with different ranges of specific bioactivities.     

Studies on lectins XXXV. Water-soluble O-glycosyl polyacrylamide derivatives for specific precipitation of lectins

By copolymerization of acrylamide and allyl glycosides of various sugars, O-glycosyl derivatives of polyacrylamide copolymers were prepared. The sugar content of the copolymers can be varied in the range 0–40%, their sedimentation coefficient shows the values of 2.5–2.7 S; the molecular weight of an $\text{O-α-}\text{d}\text{-}\text{mannopyranosyl}$ polyacrylamide copolymer (29% mannose, s_20,w⁰ = 2.9 S) was estimated as 44 500. Copolymers with incorporated glycosyl residues interacting specifically with lectins yield precipitates with them upon immunodiffusion in cellulose acetate. The quantitative precipitin curves obtained with these copolymers are similar to those produced by quantitative precipitation of lectins with natural polysaccharides. The copolymers may serve as model substances of natural polysaccharides.     

Multiple forms of glucocorticosteroid receptors in the neural retina of chick embryo,revealed by polyacrylamide gel electrophoresis

The glucocortiocoid receptors in the cytosol of neural retina of the 15-day chick embryo were analyzed by quantitative polyacrylamide gel electrophoresis. Maintenance of the triamcinolone acetonide (TA)-receptor complexes under conditions of electrophoretic analysis is dependent on temperatures not exceeding ?2 °C and is favored by low ionic strength, but is relatively insensitive to changes in pH between 5 and 10. Polyacrylamide gel electrophoresis in highly crosslinked Resolving Gels (15% crosslinking with N,N′-diallyltartardiamide) at low wattage and under temperature control at ?2 °C, allowed for detection and partial characterization of over 80% of the specific TA-binding activity of the tissue. One form of the glucocorticoid receptor, designated as complex II, was found to have a molecular weight (M_r) of 175,000. In addition, specifically bound TA was found in a multimillion M_r aggregate which was unable to enter gels of any concentration investigated and has been designated TA-complex I. The ratio of complex I/complex II increased with increasing gel concentration, indicating physical or chemical interaction between II and I. A polyacrylamide gel electrophoresis rerun of isolated TA-complex II gave rise to two smaller TA-binding species: Component B, of M_r 108,000 and component A, a relatively fast migrating molecule which could not be characterized under the conditions used. The ratio of $\text{B}\text{A}$ appeared constant and close to 2, suggesting that A and B may be significant structural elements of complex II. Polyacrylamide gel electrophoresis of isolated TA-complex I gave rise to component C of M_r 60,000, but not to components A or B. Components A and B associated to a large M_r complex, designated as I′, which was revealed to an extent directly proportional to gel concentration. Similarly, component C aggregated to I″, as evidenced at elevated gel concentrations. In conclusion, it has been possible to define by gel electrophoresis three of the molecular species (A, B, and C) that comprise the glucocorticoid receptor, and the possible relationships between them.     

Synthesis of thermo-sensitive polyacrylamide derivatives for affinity precipitation and its application in purification of lysozyme

The thermo-sensitive N-alkyl substituted polyacrylamide polymer was synthesized by radical polymerization and its lower critical solution temperature (LCST) was controlled to be 28 °C. The thermo-sensitive recovery of polymer was over 95% in the presence of 0.05 M NaClO₄. Cibacron Blue F3GA was covalently immobilized onto the polymer via the nucleophilic reaction between the active chlorine atom of its triazine ring and the hydroxyl group of the polymer. The ligands density was 30 μmol g⁻¹ polymer. The adsorption capacity of lysozyme on the polymer was 3.4 mg g⁻¹polymer in affinity precipitation process. And over 90% of adsorbed lysozyme was eluted by 0.5 M KSCN at pH 8.0. When the affinity polymer was applied in the purification of lysozyme from egg white, the purification factor was 28 and lysozyme yield was 80% or so.     

The electrophoretic mobility of double-stranded RNA in polyacrylamide gels as a function of molecular weight.

A re-evaluation of the mobility of double-stranded RNA on polyacrylamide gels over a molecular weight range of 0.46-6.3 . 10(6) was carried out using double-stranded RNAs of: bacteriophage ?6; virus like particles or mycoviruses of Penicillium chyrsogenum, Penicillium stoloniferum and Helminthosporium maydis, and reovirus type III. When the relative mobility on polyacrylamide gels was plotted as a function of log molecular weight, a smooth curve could be drawn which passed through all points. The implications of these findings to the determination of molecular weight of double-stranded RNA by polyacrylamide gel electrophoresis are discussed.     

Protein Engineering of Wzc To Generate New Emulsan Analogs

Acinetobacter venetianus Rag1 produces an extracellular, polymeric lipoheteropolysaccharide termed apoemulsan. This polymer is putatively produced via a Wzy-dependent pathway. According to this model, the length of the polymer is regulated by polysaccharide-copolymerase (PCP) protein. A highly conserved proline and glycine motif was identified in all members of the PCP family of proteins and is involved in regulation of polymer chain length. In order to control the structure of apoemulsan, defined point mutations in the proline-glycine-rich region of the apoemulsan PCP protein (Wzc) were introduced. Modified wzc variants were introduced into the Rag1 genome via homologous recombination. Stable chromosomal mutants were confirmed by Southern blot analysis. The molecular weight of the polymer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five of the eight point mutants produced polymers having molecular weights higher than the molecular weight of the polymer produced by the wild type. Moreover, four of these five polymers had modified biological properties. Replacement of arginine by leucine (R418L) resulted in the most significant change in the molecular weight of the polymer. The R418L mutant was the most hydrophilic mutant, exhibiting decreased adherence to polystyrene, and inhibited biofilm formation. The results described in this report show the functional effect of Wzc modification on the molecular weight of a high-molecular-weight polysaccharide. Moreover, in the present study we developed a genetic system to control polymerization of apoemulsan. The use of selective exogenous fatty acid feeding strategies, as well as genetic manipulation of sugar backbone chain length, is a promising new approach for bioengineering emulsan analogs.     

The partition of glycosaminoglycan-quarternary ammonium complexes II. The effects of polymer molecular weight and sulfation

Previous results have shown the possibility for obtaining high-resolution separations of glycosaminoglycans by partition in butanol/aqueous two-phase systems containing quarternary ammonium salts. In this paper, the effects on partition behavior of both polymer molecular weight and sulfation were examined. Two series of fractioned chondroitin sulfate polymers were isolated in which the molecular weight and sulfation varied systematically. In the molecular weight series the six samples, spanned the range from 3200 ± 300 to 19 700 ± 500 and each sample carried 0.8 sulfate groups per uronic acid residue. In the sulfation series, each sample had an essentially constant molecular weight of 13 000, but the sulfation varied from 0.58 to 0.88 sulfate groups per uronic acid. The $\text{C}{}_{50}{}^1$ of each of these samples was determined in the 1-butanol/aqueous NaCl phae system containing 1% hexadecylpyridinium chloride. In the series wherein the molecular weight varied, the C₅₀ increased with molecular weight up to 12 000 where a limiting value was reached. In the series wherein the sulfation varied, a linear relationship was found between the C₅₀ and the square of the number of anionic substituents per disaccharide. These results show that fractionation by partition techniques will be sensitive to the anionic nature of the polymer, but for the common connective tissue glycosaminoglycan, there will be no fractionation according to molecular weight.     

Electrophoresis of proteins in semidilute polyethylene glycol solutions: Mechanism of retardation

The retardation of proteins in the M_t range of 15–500 kDa in capillary electrophoresis conducted in semidilute solutions of the polymer polyethylene glycol (M_t range 0.2–8.0 × 10⁶), was measured. The purpose was to test the predictions of the scaling theory with regard to the relation of retardation to (a) the M_t of the polymer, (b) the concentration of the polymer, and (c) the radius of the protein particles. These predictions derive from a mechanism that relates retardation to the screening length of the polymer solution, viewed as the average distance between the entanglement points of polymer chains. For the molecular weight range from 60 to 500 kDa of (near) spherical proteins, the retardation was found to be related to polymer concentration c asμ/μ_o = exp(-Ac^0.69)where μ/μ₀ is the retardation expressed as the ratio between the mobility in polymer solution and that in free solution. The value of the exponent of 0.69 is in close agreement with the value of 0.75 predicted by the scaling theory. Parameter A was found (a) to scale as the 0.04th power of M_t (polymer), approximating the predicted value of 0; and (b) to be proportional to particle radius as predicted. All measured values of retardation were independent of electric field strength in the range of 37–370 V/cm. Thus, experimental findings are consistent with the mechanism relating electrophoretic retardation to the screening length of the polymer network in the specified molecular weight range of proteins. Under the same conditions, log(μ/μ₀) of proteins with M_t's less than 60 kDa (a) scales as the −0.06th power of M_t (polymer), and (b) is proportional to polymer concentration, suggesting a retardation mechanism that is not related to the screening length. © 1997 John Wiley & Sons, Inc. Biopoly 42: 183–189, 1997