Chemical pattern formation in gels

Precipitate forming chemical reactions have been studied in chemically crosslinked poly(vinyl alcohol) gel medium. One of the reactive components was incorporated into the gel, the other was allowed to diffuse into the swollen network. This reaction-diffusion process often results in pattern formation which can occur intermittently in terms of time and space or both. Experiments were carried out in order to investigate the effect of various factors (crosslinking density, swelling degree as well as the concentrations of the outer and inner electrolyte). The following precipitates were intensively studied: Mg(OH)₂, Cr(OH)₃, PbCl₂. Three basically different morphologies (Liesegang band formation, moving bands and tree-like structure) have been observed and investigated. One- and twodimensional computer simulations were carried out in order to establish the influence of various factors on the precipitation.     

Native protein mapping and visualization of protein interactions in the area of human plasma high‐density lipoprotein by combining nondenaturing micro 2DE and quantitative LC‐MS/MS

A human plasma sample was subjected to nondenaturing micro 2DE and a gel area (5 mm × 18 mm) that includes high‐density lipoprotein (HDL) was cut into 1 mm × 1 mm squares, then the proteins in the 90 gel pieces were analyzed by quantitative LC‐MS/MS. Grid‐cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC‐MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data. Totally 154 proteins were assigned in the 90 gel pieces and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The map of apolipoprotein (Apo) A‐I showed a wide apparent mass distribution characteristic to HDL and was compared with the maps of the other 153 proteins. Eleven proteins showed maps of wide distribution that overlapped with the map of Apo A‐I, and all have been reported to be the components of HDL. Further, seven minor proteins associated with HDL were detected at the gel positions of high Apo A‐I quantity. These results for the first time visualized the localization of HDL apolipoproteins on a nondenaturing 2DE gel and strongly suggested their interactions.     

Analysis of protein/polypeptide interactions in human plasma using nondenaturing micro-2-DE followed by 3-D SDS-PAGE and MS

Previously, we have reported on the analysis of human plasma proteins on a nondenaturing micro-2-DE (mu2-DE) gel, using in-gel digestion followed by MALDI-MS and PMF [1]. Many of the spots on the mu2-DE gel showed apparent masses much larger than the calculated masses of their assigned polypeptides, suggesting noncovalent or covalent interactions between the polypeptides. In the present study, we aimed to further analyze the plasma protein spots on a nondenaturing mu2-DE gel, on which protein/polypeptide interactions have been suggested. The proteins in the spots were extracted under alkaline conditions and subjected to 3-D separation using SDS-PAGE in microslab gel format (muSDS gel) with or without the sample treatment of reduction-alkylation. The clear bands in each lane of the muSDS gels demonstrated the successful extraction of proteins from the relevant gel spot and visualized the relative contents of the polypeptides in the spot. Most of the bands were assigned by in-gel digestion followed by MALDI-MS and PMF (MASCOT/Swiss-Prot). The large discrepancy between the apparent mass value of a protein spot and the estimated mass values of the polypeptide bands on a nonreducing muSDS gel strongly suggested noncovalent polypeptide interactions. The differences in the polypeptide separation patterns on the muSDS gels, between with and without the treatment of reduction-alkylation, confirmed polypeptide disulfide bonding. The method employed here, aiming to integrate information on the proteins separated on nondenaturing 2-DE gels with that on the interactions between polypeptides, would help the comprehensive understanding of complex protein systems.     

Electrophoretic separation of protein kinases: altered mobility with different crosslinking agents in the presence of certain detergents

Nondenaturing polyacrylamide gel electrophoresis was used to separate protein kinases in crude extracts and subcellular fractions of murine erythroleukemic cells. The kinases were detected using an in situ phosphorylation assay. The electrophoretic patterns obtained using gel bound to GelBond and prepared with AcrylAide differed from those seen without GelBond and with N,N'-methylenebisacrylamide as cross-linker. In an attempt to improve the resolution of the bands in the membrane fractions, detergent-treated preparations were electrophoresed through gels which contained either 0.1% Triton X-100 or 0.1% Nonidet P-40. The resolution of the bands in this fraction was not, however, improved with the inclusion of the nonionic detergent in the gels. When cytosol was electrophoresed through gels containing detergent, a major band of cAMP-dependent protein kinase activity showed a marked shift in mobility. This may have been the result of a structural change, altering the shape and possibly affecting the charge on the molecule, or the enzyme may have formed aggregates with the detergent.     

Patterns with high rhythmicity levels in multicomponent Liesegang systems.

Liesegang banding is the display of rhythmic strata of precipitate as co-precipitate ions interdiffuse in a gel medium. Complex periodic patterns as well as aperiodic structures could emerge, notably in systems where more than one salt is precipitated. The use of three cations (Co2+) Ni2+, and Mg2+) in the banded precipitation of their hydroxides resulted in an unusual pattern with a consistently increasing rhythmicity. A periodic structure marked by the succession of band multiplets with increasing number of bands (from singlets to doublets to triplets to quadruplets, consistently) was observed. Such rhythmic patterns are obtained as the initial Mg2+ concentration ([Mg2+]0), chosen as a control parameter, increases through a critical value. At high [Mg2+]0, the trend breaks after a long time elapses. Two types of bifurcation are therefore experienced by such a system: concentration bifurcation and diffusive (time/space) bifurcation. The dynamics is elucidated on the basis of an analysis of the bands in certain groups, and gel regions between these groups, as well as between group blocs (here, a bloc denotes a succession of multiplet groups, with repetitively the same number of bands). Finally, similarities between our system and naturally occurring rhythmic patterns are emphasized and discussed.     

Detection of errors in dinucleotide repeat typing by nondenaturing electrophoresis.

This study shows that consideration of minor bands (heteroduplex, shadow, and faint bands) associated with allele bands in nondenaturing polyacrylamide gel electrophoresis (PAGE) after polymerase chain reaction (PCR) is effective for detecting PCR processing errors that lead to mistyping of heterozygotes as homozygotes. Notably, we show that minor bands in native gels are highly effective for detecting allele dropout and preferential amplification in PCR amplification of dinucleotide repeats. These findings are based on an analysis of Mendelian inheritance patterns in families, and the reproduction of heterozygous band patterns by mixing homozygous DNAs before PCR, for a total of six (AC)n repeats located on human chromosome 11p15. To investigate the utility of our approach, a large population sample of 405 unrelated individuals was genotyped for each (AC)n repeat using minor bands as internal quality controls. Genotype frequencies at each of the six loci were in close agreement with Hardy-Weinberg proportions, which suggests that there were few genotyping errors. Our observations add to the evidence indicating that minor bands in native gels are of diagnostic value in the genotyping of dinucleotide repeats.     

Comparison of oligoclonal immunoglobulin G bands in multiple sclerosis cerebrospinal fluid by immunoblotting and immunofixation

We previously analyzed unconcentrated cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and other neurologic diseases by isoelectric focusing in agarose gel. We have now developed an immunoblot method for detection of oligoclonal IgG bands in unconcentrated MS CSF. The oligoclonal IgG band patterns seen after immunoblotting were compared with those of conventional immunofixation. Although immunoblotting was found to be rapid the resolution and intensity of oligoclonal IgG bands were somewhat better after immunofixation. Since immunofixation is simpler than immunoblotting, we recommend that clinical laboratories use immunofixation after isoelectric focusing to detect oligoclonal IgG bands in CSF.     

Computerized methods for analyzing two-dimensional agarose gel electropherograms

Previous methods interpret zonal or polydisperse gel patterns of two-dimensional Serwer-type gels in terms of size and free mobility (surface net charge density). These two parameters have been determined for each component without quantitatively measuring the abundance of the components. The present study advances these previous methods by determining the relative concentration of each component by computer evaluation of densitometrically analyzed gel patterns. Suitable procedures and their underlying algorithms are presented. The mathematical routines are implemented in a user-friendly software package, called GelFit and designed for a Macintosh personal computer. The program input consists of digitized images of gel staining patterns exemplified by those obtained from electrophoresis of native subcellular-sized particles. The data are processed through the following steps: (i) Noise reduction and calibration. (ii) Geometrical transformation of the pattern onto a rectangular size/free mobility coordinate system using rationales of the extended Ogston model. (iii) Analysis of the transformed image to determine density maxima, density profiles along iso-free-mobility or iso-size lines, curve fitting of one-dimensional profiles or two-dimensional surfaces using Gaussian functions and curve stripping of surfaces to determine the possible number of particle populations.     

Electrophoretic separation of gold nanoparticles according to bifunctional molecules‐induced charge and size

Gold nanospheres modified with bifunctional molecules have been separated and characterized by using agarose gel electrophoresis as well as optical spectroscopy and electron microscopy. The electrophoretic mobility of a gold nanosphere capped with 11‐mercaptoundecanoic acid (MUA) has been found to depend on the number of MUA molecules per gold nanosphere, indicating that it increases with the surface charge of the nanoparticle. The extinction spectrum of gold nanospheres capped with MUA at an MUA molecules per gold nanosphere value of 1000 and connected via 1,6‐hexanedithiol (HDT) decreases by 33% in magnitude and shifts to the red as largely as 22 nm with the increase of the molar ratio of HDT to MUA (R_HM). Gold nanospheres capped with MUA and connected via HDT have been separated successfully using gel electrophoresis and characterized by measuring reflectance spectra of discrete electrophoretic bands directly in the gel and by monitoring transmission electron microscope images of gold nanoparticles collected from the discrete bands. Electrophoretic mobility has been found to decrease substantially with the increment of HDT to MUA, indicating that the size of aggregated gold nanoparticles increases with the concentration of HDT.     

Detection of protease activities using specific aminoacyl or peptidyl p-nitroanilides after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and its applications

A general method for detecting protease activities on acrylamide or agarose gels after sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) using specific aminoacyl p-nitroanilide (NA) or peptidyl NA as substrate is described. This method is extended from the spectrophotometric assay of p-nitroaniline, which is a chromogenic product liberated by protease action on aminoacyl NA or peptidyl NA. The acrylamide gel containing protein bands was dipped directly into a solution which contained specific synthetic aminoacyl NA or peptidyl NA as a substrate or had been overlaid with an agarose gel containing the same substrate. The p-nitroaniline released on the acrylamide or agarose gel by the specific protease was diazotized with sodium nitrite and then coupled to N-(1-naphthyl)-ethylenediamine to produce distinct activity band(s). The substrates used for protease activity staining on gels were identical to those used for spectrophotometric assays. Some applications are described.     

Controlling and engineering precipitation patterns

Controlling and engineering chemical structures are the most important scientific challenges in material science. Precipitation patterns from ions or nanoparticles are promising candidates for designing bulk structure for catalysis, energy production, storage, and electronics. There are only a few procedures and techniques to control precipitation (Liesegang) patterns in gel media (e.g., using an electric field, varying the initial concentration of the electrolytes). However, those methods provide just a limited degree of freedom. Here, we provide a robust and transparent way to control and engineer Liesegang patterns by varying gel concentration and inducing impurity by addition of gelatin to agarose gel. Using this experimental method, different precipitation structures can be obtained with different width and spatial distribution of the formed bands. A new variant of a sol-coagulation model was developed to describe and understand the effect of the gel concentration and impurities on Liesegang pattern formation.     

三方硒电子结构的EH研究

计算了三螺旋硒链的一维能带, 三方硒的三维能带和Se_6簇的能级。能带分为三组: Se-Se成键带, 孤对电子带和Se-Se反键带。符合Se-Se, 成键的价电子层结构。同时对电子态进行了空间群的对称性分类。为了更广泛的将固体的计算结果与原子簇做比较, 定义了簇轨道重叠布居, 它是原子簇计算态密度的轨道重叠布居权重值。计算表明, Se_6簇(D_(3d))的态密度和簇轨道重叠布居分别与三方硒的态密度和晶体轨道重叠布居颇为类似, 说明两者成键本质的类似性。带隙和态密度与相应的实验数据作了比较。     

A rapid gel electrophoretic chip for serum cholesterol determination

We present a rapid gel electrophoretic chip, composed of 2.5% (w/v) acrylamide and 1% (w/v) agarose gel, for serum cholesterol determination using a photo lithography technique. After optimizations, we determined the lipoprotein concentration of standard serum using a conventional enzyme method. The serum was diluted, stained and loaded for 15 min onto the chip. After loading, the intensities of low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) bands separated at the chip were estimated using an image analyzer. The intensities of these bands corresponded to concentrations obtained from a standard enzyme-based method. The detected LDL-C and HDL-C concentrations were linear up to 146 mg dL(-1) and 53 mg dL(-1) respectively. Finally, we carried out the cholesterol analysis using real biological samples obtained from nine volunteers using our electrophoretic chip. The LDL-C and HDL-C levels detected using our chip correlated well with the results obtained using the conventional enzyme-based method r(2) = 0.98 and r(2) = 0.86 for LDL-C and HDL-C, respectively. Although our sample size is small and confined only to health volunteers, we have demonstrated that this proof-of-concept gel electrophoretic chip can determine lipoproteins, simultaneously.     

The distribution of particles characterized by size and free mobility within polydisperse populations of protein-polysaccharide conjugates, determined from two-dimensional agarose electropherograms

New approaches for the characterization of polydisperse particle populations are presented*. The investigated samples contain virus-sized protein-polysaccharide conjugates which had previously been prepared as immunogens against bacterial meningitis (Hib). The analysis is based on two-dimensional agarose electrophoresis (Serwer-type). This method, like the one of O'Farrell, achieves a separation according to size and charge. It relies on a different principle, however, and is applicable to nondenatured particles which are 100 to more than 1000 times larger in mass than regular uncrosslinked proteins. Data from stained gel patterns are evaluated by the computer program ELPHOFIT, which makes it possible to standardize the gel and to construct a nomogram which defines every position on the gel in terms of particle size and free mobility (related to surface net charge density). The output of ELPHOFIT, consisting of nomogram parameters, is transferred to the image processing program GELFIT. This software is used to evaluate the computer images obtained by digitizing the stained gel patterns: (i) The nomogram is electronically superimposed on the computer image. (ii) The gel pattern is transformed from a curvilinear to a rectangular coordinate system of particle size and free mobility. The center of gravity as well as density maxima are given in coordinates of particle size and free mobility. Ranges of grey levels can be accentuated by adding 16 pseudocolors. (iii) Using surface-stripping techniques, GELFIT provides an estimate for the number of major subpopulations within each preparation. (iv) Numerical values for the distribution of particle size and free mobility are determined. Using program IMAGE, the quantitative physical assessment of a given conjugate preparation is presented in the form of a computer-generated three-dimensional plot, the shape of which serves to identify and characterize the preparation visually. The data analysis based on digitized two-dimensional gel patterns is automated to an extent that a technician can perform routine evaluations. It uses the Macintosh II personal computer.     

Requirements for the application of protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis and randomly amplified polymorphic DNA analyses to product speciation

Raw, cooked, fried, smoked and gravad (brine-cured) products were analyzed by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins and by randomly amplified polymorphic DNA (RAPD) in order to identify the species used in their manufacture. The discriminatory power of SDS-PAGE was dependent primarily on the composition and secondarily on the size of the gels: the Laemmli buffer system with 15% acrylamide and 0.087% piperazine diacrylamide separating gels resolved more discriminant protein bands than any of the commercial gels tested. Some of the processing conditions induced alterations in the protein patterns that made identification dubious. Differentiation even between closely related species was easier by RAPD than by SDS-PAGE. Neither the processing conditions nor the tissue from which the DNA was extracted had a significant effect on the RAPD profiles. For identifications based on SDS-PAGE, one should use an optimized gel composition and separate the sample under analysis in the same gel as the references. For RAPD-based identifications, the unknown sample should be amplified together with reference samples and separated in the same gel.     

Discontinuous electrophoresis of beta-caseins for the determination of bovine caseins in milk and dairy products.

Discontinuous acidic anodic polyacrylamide gel electrophoresis enables the separation of bovine beta-caseins from those of ovine and caprine. Interfering protein bands as a consequence of ripening or processing have not been detected. After evaluation of the stained gel by laser densitometry, quantification was performed with calibration standards on the same gel by the ratio of the peak areas from bovine to ovine and bovine to caprine, respectively. Thus, independence from the extractability of proteins affected by denaturation and ripening (which might in some cases raise the limit of detection) is achieved. The range of quantification extends from 5 to approximately 70% bovine casein in relation to total casein.     

Multiple forms of aldolase in organ extracts of the rat. On aldolases, 8

The aldolase activities of crude rat tissue extracts were separated by starch gel electrophoresis. Aldolase was located on the gel by a specific staining method. Nine bands of different electrophoretic mobility were stainable by this technique. Organ specific isoenzyme patterns could be demonstrated.     

Native multimer analysis of plasma and platelet von Willebrand factor compared to denaturing separation: implication for the interpretation of satellite bands

Blue native electrophoresis (BNE) was applied to analyze the von Willebrand factor (vWF) multimers in their native state and to present a methodology to perform blue native electrophoresis on human plasma proteins, which has not been done before. The major difference between this method and the commonly used SDS-agarose gel electrophoresis is the lack of satellite bands in the high-resolution native gel. To further analyze this phenomenon, a second dimension was performed under denaturing conditions. Thereby, we obtained a pattern in which each protein sub-unit from the first dimension dissociates into three distinct sub-bands. These bands confirm the triplet structure, which consists of an intermediate band and two satellite bands. By introducing the second dimension, our novel method separates the triplet structure into a higher resolution than the commonly used SDS-agarose gel electrophoresis does. This helps considerably in the classification of ambiguous von Willebrand's disease subtypes. In addition, our method has the additional advantage of being able to resolve the triplet structure of platelet vWF multimers, which has not been identified previously through conventional SDS-agarose electrophoresis multimer analysis. This potential enables us to compare the triplet structure from platelet and plasmatic vWF, and may help to find out whether structural abnormalities concern the vWF molecule in the platelet itself, or whether they are due to the physiological processing of vWF shed into circulation. Owing to its resolution and sensitivity, this native separation technique offers a promising tool for the analysis and detection of von Willebrand disorder, and for the classification of von Willebrand's disease subtypes.     

Detection of PEGylated proteins in polyacrylamide gels by reverse staining with zinc and imidazole salts

The reverse staining, with imidazole-SDS-zinc, of PEG-linked proteins separated by SDS-PAGE was studied. Using model conjugates (interferon-alpha 2b (IFN-alpha2b) reacted with either a branched-chain (40,000) PEG (PEG2,40) or a linear monomethoxy PEG polymer (Mr of 12,000) and chromatographically purified monoPEG2,40-IFN-alpha2b), conventional small-format analytical gels (<1 mm thick) showed typical detection patterns (i.e., transparent, colorless bands clearly discernible against a zinc imidazolate-generated white gel background), in less than 20 min. Nonreacted (free) PEG was almost undetected, as expected. The reverse-stained PEGylated IFN-alpha2b patterns were qualitatively indistinguishable from those of parallel gels stained with iodine (I2). The LOD was estimated in the low nanogram range (e.g., at about 7 ng for mono- or bi-PEG2,40 IFN-alpha2b per lane on gradient (4-17%) gels). Also, this stain allowed the visualization of Coomassie blue-undetected PEG-IFN bands, and could be restained with I2. PEGylated species of lysozyme, a low-molecular-weight peptide, ovalbumin, and chymotrypsin were used to demonstrate the generality of this stain. We also show (i) how to counteract the adverse effect of some parameters (e.g., gel thickness above 1 mm, long gel length, low (e.g., 4-6%) acrylamide concentration) on the reverse staining process and (ii) that the properties of the reverse-stained PEGylated proteins remain unchanged, as judged by analyzing both the ion exchange chromatography-based positional isomer separation profile and enzyme-linked immunosorbent response of PEG-IFN recovered from gels. Consequently, this technique may be useful for the rapid analysis or the small-scale preparation of PEGylated proteins.     

Density gradient electrophoresis of cells in a reversible gel

Density gradient electrophoresis permits the separation of cell types according to surface charge density with high resolution. Any source of flow compromises the resolving power of density gradient electrophoresis. Although procedures have been devised to successfully counteract electroosmotic and convective flows, the final collection of separands requires that they be pumped out of the electrophoresis column. Experiments were therefore designed to test the hypothesis that this flow could also be eliminated by trapping the separated bands in a gel, from which they could be collected by slicing the gel cylinder. Glutaraldehyde-fixed rat and rabbit erythrocytes were used as test particles in a phosphate-buffered isotonic Ficoll-sucrose density gradient in a 2.2 cm diameter, thermostated vertical glass column that could be opened at both ends. Two types of agarose were used as gel polymers: Electrophoresis grade agarose (J.T. Baker Chemical Co.) at final concentrations of 0.1 to 0.25% and SeaPrep ultralow gelling agarose (Marine Colloids Div., FMC Corp.) at a final concentration of 1.0%. Electrophoretic separability of the test particles and fluid stability were tested independently at 55 degrees C and 32 degrees C at which the two agaroses were, respectively, liquid. The experiments demonstrated that the higher temperatures required and the presence of agarose compromised neither the stability of the density gradient nor the migration properties of the cells, and cells can be separated in a sol at a temperature that is compatible with cell viability.