Determination of the molecular weight of proteins by electrophoresis in slab gels with a transverse pore gradient of crosslinked polyacrylamide in the absence of denaturing agents

The molecular weight of proteins under nondenaturing conditions can be determined through polyacrylamide electrophoresis by comparing their relative mobilities at different gel concentrations with the relative mobilities of standard proteins under the same conditions (J. L. Hedrick and A. J. Smith (1968) Arch. Biochem. Biophys. 126, 155). This work describes a procedure that eliminates the need for several gels of different acrylamide concentrations with the use of a slab gel with a transverse pore gradient of crosslinked polyacrylamide.     

Field inversion gel electrophoresis in denaturing polyacrylamide gels.

The velocities of single stranded DNA molecules in denaturing polyacrylamide gels during symmetric and asymmetric field inversion were measured at different pulse times and gel concentrations. Under the conditions chosen in our study, pulse times as short as a few milliseconds lead to a retardation of DNA molecules larger than 400 bases. We found that a field inversion with an electric field in the forward direction of about double the strength of that applied in the backward direction is a good compromise between the degree of retardation, the temperature control requirements and the run time of the gel.     

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.     

Detection of laccase activity in polyacrylamide gels after electrophoresis under denaturing conditions

Summary Specific laccase activity was detected on SDS-PAGE using 2,2-Azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid), guaiacol and syringaldazine as substrates. Enzyme activity was detected immediately following electrophoresis, after the detergent diffused from the gel into a renaturation buffer and subsequent Coomassie blue staining. Identification of laccase in a protein mixture and estimation of its molecular weight were done simultaneously.     

Two-dimensional electrophoresis of plasma proteins without denaturing agents.

A technique of two-dimensional polyacrylamide gel electrophoresis for the separation of plasma proteins is described. Human plasma proteins were separated by isoelectric focusing followed by electrophoresis in a 4 to 21% linear gradient gel slab. No denaturing agent was used throughout the procedure, so that the analysis of native proteins is possible. Two-dimensional patterns obtained from normal human plasma samples were recorded as "staining density maps," which are similar to contour line maps, and more than 230 protein spots were counted reproducibly on each "staining density map." This technique permits the simultaneous estimation of pI's and approximate molecular weights of native proteins on the slab gel. Applications of this technique to an IgA myeloma plasma sample and a porcine serum sample are described.     

Molecular-weight determination of animal-cell RNA by electrophoresis in formamide under fully denaturing conditions on exponential polyacrylamide gels.

A method for electrophoretic analysis of RNA under fully denaturing conditions on exponential gradient polyacrylamide gels is described. Full denaturation, and strand separation of DNA - RNA hybrids and double-stranded RNA is obtained in dry formamide only if electrophoresis is carried out at 45 degrees and 55 degrees C, respectively. In such conditions, the effects of secondary structure of RNA, important in aqueous medium, are suppressed and a linear correlation is obtained between the logarithm of the molecular weight of an RNA and its final position in the gel over the entire molecular weight range of 10(4) - 10(7). Based on absolute molecular weight standards, obtained from sequenced rRNA of Escherichia coli and tRNA and extrapolating to higher molecular weights the size of animal cell was reexamined. Precursor tRNA from HeLa cells migrates according to a molecular weight of 4.1 x 10(6). Nascent precursor mRNA has molecular weights of up to 5 x 10(6) in the case of duck erythroblasts and of up to 10(7) in HeLa cells. This seems to represent the largest size of non-viral animal-cell RNA molecules.     

Detection of DNA-binding proteins in polyacrylamide gel after denaturing electrophoresis]

A simple method for detection of DNA-binding proteins is offered. These proteins can be revealed, following their electrophoretic separation in sodium dodecyl sulfate (SDS)-polyacrylamide gel containing labeled DNA, by washing the gel in buffer to remove SDS and to allow protein renaturation. Protein-free DNA is washed out, remaining in the DNA-binding proteins that restored their original characteristic. After autoradiography these proteins are seen as black bands (by one-dimensional gel electrophoresis) or spots (by two-dimensional gel electrophoresis) on a grey background. High sensitivity of the method is shown by using protein fractions of rat liver and a standard method.     

Partitioning and diffusion of proteins and linear polymers in polyacrylamide gels.

The equilibrium partition coefficient (K) and diffusion coefficient (Dgel) of two proteins and two linear polymers were measured as a function of polymer content of a 2.7% cross-linked polyacrylamide (PA) gel. The gel concentration, expressed as a volume percentage of PA in the gel (phi), varied between 0 and 14%. The measurements were made by fluorescence spectroscopy; fluorescent dyes were covalently attached to the macromolecules. The dependence of K on phi for the proteins agrees with a model of the gel network as randomly placed, impenetrable rods. The diffusion data are interpreted in terms of an effective medium theory for the mobility of a sphere in a Brinkman fluid. Using values of the Brinkman parameter in the literature, the effective medium model with no adjustable parameters fits the diffusion data for the proteins very well but underpredicts Dgel for the linear polymers. The gel effect on partitioning is significantly greater than that on diffusion. The permeability (KDgel) of bovine serum albumin decreased by 10(3) over the range phi = 0 --> 8%, and the ratio of permeabilities for ribonuclease compared to BSA increased from 2 to 30.     

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.     

A method for separating DNA fragments by electrophoresis in polyacrylamide concentration gradient slab gels

Electrophoresis on slab gels containing a linear gradient of polyacrylamide concentration has been used to separate DNA fragments obtained by restriction of viral DNAs. A simple method of preparing gradient gels using a sucrose density-gradient mixer and preexisting slab gel apparatus is described. DNA fragments of molecular weights 7 × 10⁴–14 × 10⁶ have been fractionated on gels of 3.5–7.5% and 2.5–7.5% acrylamide concentration. In addition to the wide range of fragment sizes which may be run on a single gel, a further advantage of the system is that much sharper bands are obtained compared to conventional constant concentration gels, thus improving resolution.In the molecular-weight range below 5 × 10⁶, for bands whose terminal velocities in the polyacrylamide concentration gradient approach zero, an approximately linear relationship holds between the logarithms of the molecular weights of the fragments and the logarithms of the distances they have migrated in the gel. Thus, by choosing a suitable upper limit to the concentration gradient, the gel system provides a method for estimating approximate molecular weights of unknown DNA fragments, by comparing their mobilities to known standards.     

Ultramicrodetection of proteins in polyacrylamide gels.

Here we report the development of a highly sensitive procedure to detect proteins within separation matrices which should facilitate the characterization of rare proteins. The procedure is based on photochemical reactions where very low amounts of silver are deposited around proteins and in a series of steps are converted to silver sulfide. When this conversion is carried out in the presence of [35S]thiourea the resulting radioactive silver sulfide allows detection down to femtogram quantities of protein. In this work we applied the above principle to proteins separated on sodium dodecyl sulfate-polyacrylamide gels, thus not influencing physical and chemical parameters which are important for separation. This procedure should find application in any technique where detection of very low or limited amounts of proteins are required.     

Molecular weight determinations of proteins by polyacrylamide gel electrophoresis with sodium dodecyl sulfate in just the sample solution

This report describes a simple modification of the procedure for SDS polyacrylamide gel electrophoresis by using SDS in the sample solution and eliminating its use in the gel and the electrode compartments. The results obtained were comparable to those with SDS in the entire system. The log molecular weight-relative mobility plots for a variety of proteins, the reproducibility and the appearance of the bands such as their intentisy and sharpness were very similar. Therefore SDS in the entire system is not necessary to determine the molecular weight of a protein.     

Analysis of collagen cyanogen bromide peptides using electrophoresis in continuous concave gradient polyacrylamide gels.

A rapid and simple spectrophotometric method is described for the estimation of microgram quantities of glycosaminoglycans following the formation of soluble complexes with alcian blue dye. The method is based on the different absorption spectra of the dye and dye-glycosaminoglycan complexes. No heating, centrifugation, lengthy equilibration, or sophisticated instrumentation which hamper other methods are required. Samples are mixed with freshly prepared dye solution and absorbance readings at 480 nm are compared to an appropriate standard curve. Albumin and individual monosaccharides do not interfere with the assay but high concentrations of chloride ion do. The method is suitable for the estimation of total glycosaminoglycan levels in biological fluids such as urine and blood.