Capillary isoelectric focusing immunoassay as a new nanoscale approach for the detection of oligoclonal bands

The detection of oligoclonal bands (OCBs) in cerebrospinal fluid is an indicator of intrathecal synthesis of immunoglobulins which is a neurochemical sign of chronic inflammatory brain diseases. Intrathecally synthesized IgGs are typically observed in patients with multiple sclerosis. The current standard protocol for the detection of OCBs is IEF on agarose or polyacrylamide gels followed by immunoblotting or silver staining. These methods are time consuming, show substantial interlaboratory variation and cannot be used in a high throughput‐approach. We have developed a new nanoscale method for the detection of OCBs based on automated capillary IEF followed by immunological detection. Evidence for intrathecal IgG synthesis was found in all tested patients (n = 27) with multiple sclerosis, even in two subjects who did not have oligoclonal bands according to standard methods. The test specificity was at 97.5% (n = 19). Our findings indicate that the novel OCB‐CIEF‐immunoassay is suitable for the rapid and highly sensitive detection of OCBs in clinical samples. Furthermore, the method allows for a higher sample throughput than the current standard methods.     

Agarderivatives for chromatography, electrophoresis and gel-bound enzymes. IV. Benzylated dibromopropanol cross-linked sepharose as an amphophilic gel for hydrophobic salting-out chromatography of enzymes with special emphasis on denaturing risks.

The preparation of benzylated covalently cross-linked Sepharose 2B is described. Such gel was analyzed for its degree of substitution, and gels with three different degrees of substitution were used in chromatographic experiments with dextranase, alpha-amylase, lactate dehydrogenase, alpha-chymotrypsin and trypsin. Yields and chromatographic patterns for different eluting systems were determined. It was found that gradients combining an increase in ethylene glycol concentration with a decrease in salt concentration gave better results than did pure salt gradients. No denaturation was observed for dextranase or alpha-amylase, but the other enzymes tested were partly denatured. The most severe denaturation was observed for lactate dehydrogenase desorbed from the highest substituted gels, although the enzyme was highly active in the adsorbed state. The results and the use of amphophilic gels are discussed.     

Highly sensitive detection of ATPase activity in native gels

Native electrophoresis is a powerful tool for the separation of intact protein complexes. By incubating such gels in a suitable reaction solution, specific enzyme activities can be screened comprehensively. The recent standard procedure for determination of ATP hydrolysis activity in blue or clear native gels is based on formation of a lead phosphate precipitate. The resulting white bands are challenging for detection and documentation of low activities. For the analysis of photosynthetic ATP synthases, the method has to be adapted to deregulate the inhibition of latent ATPase functions. Therefore, we introduced an incubation of gels in detergent solution, whereby taurodeoxycholate turned out to be the most efficient activator. In order to detect low ATPase activities, a short additional incubation step subsequent to the formation of lead phosphate is recommended. By adding ammonium sulfide, the white bands are converted into brownish‐black bands of lead sulfide. Our new procedure sustains the linear quantitation range of the original lead phosphate protocol and moreover expands the detection limit.     

Differential display, subtractive hybridization, and application of methodology to search for point mutations to identify genetic defects responsible for progression in MCF10AT model of human breast disease

We describe initial studies utilizing three methods to detect differences in gene expression: (i) differential display with polyT-anchored primers; (ii) differential display with RNA arbitrarily primed polymerase chain reaction (RAP-PCR), and (iii) cDNA subtractive hybridization. Subtractive hybridization, which detects qualitative differences in gene expression, revealed no differences between a human cell line (MCF10A), originated by spontaneous immortalization of breast epithelial cells, and MCF10CA1 cells, a recently derived fully malignant, metastatic variant. The standard method of differential display with polyT anchored primers detected in excess of 100 differentially displayed bands but differential expression could seldom be verified by Northern blotting. However, RAP-PCR products frequently represent the coding region and fewer bands are detected. One gene of interest is a zinc finger protein which may be expressed more in the preneoplastic lesion-forming cells than in nonlesion-forming cells. Because most bands are not consistently differentially displayed among the variants of the MCF10 model, we suspect that point mutations rather than differential quantitative gene expression is responsible for some stage of progression. We propose that differential display of RAP-PCR products on nondenaturing gels might also detect point mutation differences.     

Affinity chromatography of lactate dehydrogenase and wheat germ lectin on new gels bearing carboxylic functions

The suitability of two new functionalized copolymer gels for use in affinity chromatography has been examined. Both gels were substituted by two ligands, one being specific for lactate dehydrogenase and the other for wheat germ lectin. The derivatives thus obtained were used successfully for the purification of two proteins with different biological activities.     

Microheterogeneity of human erythrocyte pyruvate kinase: application of immunological visualization techniques after isoelectric focusing in ultrathin gels

Immunological methods for protein visualization afford high sensitivity, good specificity and resolution. We used the indirect immunoassay with peroxidase- and 125I-labelled antibodies to investigate the microheterogeneity of pyruvate kinase from human red blood cells and can show an enzyme pattern with more than 10 single bands. Different methods were tested as to their applicability for protein fixation and subsequent immunological visualization in polyacrylamide gels. Besides the Western blot, immunofixation and ethanolic fixation can be recommended as fixation techniques, especially after isoelectric focusing in ultrathin gels.     

Detection of beta-glucanase activity on various beta-1,3 and beta-1,4-glucans after native and denaturing polyacrylamide gel electrophoresis.

beta-Glucanases were detected after polyacrylamide gel electrophoresis under native and denaturing conditions using various beta-1,3- and beta-1,4-glucans, including mixed glucans (laminarin, pachyman, carboxymethyl cellulose, lichenan and barley beta-glucan). After electrophoresis and incubation of gels, substrates incorporated into polyacrylamide gels were stained with specific fluorochromes, Sirofluor for beta-1,3 linkages and Calcofluor White M2R for beta-1,4 linkages. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. Enzymes of bacterial, fungal and plant sources could be revealed sequentially in gles containing mixed beta-(1,3)(1,4)-glucans by staining first with sirofluor followed by staining with Calcofluor White M2R. Active profiles were more diverse when substrates were stained with sirofluor. The use of purified sirofluor at pH 11.5 compared with Aniline Blue at pH 8.6 allowed better detection of beta-1,3-glucanase activities. In gels containing laminarin stained with sirofluor, bands exhibiting a more intense fluorescence than the background fluorescence were observed in addition to dark nonfluorescent bands. It is postulated that these two types of beta-1,3-glucanase activities differ by their enzymatic action (partial versus extensive hydrolysis). Analysis of fungal extracts using denaturing gels embedded with various beta-glucans displayed lysis bands migrating between 32 and 35 kDa.     

Comparison of ethanol-soluble proteins from different rye (Secale cereale) varieties by two-dimensional electrophoresis

The major storage proteins from six rye varieties, grown under the same conditions in 1997 and 1998 in Rønhave, Denmark, were analyzed by two‐dimensional (2‐D) polyacrylamide gel electrophoresis. The proteins were extracted from ground rye kernels with 70% ethanol and separated by 2‐D electrophoresis. The gels were scanned, compared using ImageMaster® software and the data sets were analyzed by principal component analysis (PCA) using THE UNSCRAMBLER software. Afterwards MATLAB was used to make a cluster analysis of the varieties based on PCA. The analysis of the gels showed, that the protein patterns (number of different proteins and their isoelectric points and molecular weights) from the six rye varieties were different. Based on the presence of unique cultivar‐specific spots it was possible to differentiate between all six varieties if the two harvest years were investigated separately. When the results were combined from the two years five varieties could be differentiated. The results from the PCA confirmed the finding of the unique spots and cluster analysis was made in order to illustrate the results. The combination of the results from 2‐D electrophoresis and other grain characteristics showed that one protein spot was located close to the parameters bread volume and bread height.     

Thermal denaturation produced degenerative proteins and interfered with MS for proteins dissolved in lysis buffer in proteomic analysis

In 1-DE, proteins were traditionally mixed with the standard Laemmli buffer and boiled for several minutes. Recently, proteins dissolved in lysis buffer were used to produce better-resolved 2-DE gels, but thermal denaturation procedure still remained in some proteomic analysis. To determine the detailed effects of thermal denaturation on SDS-PAGE and MS, both 1-DE and 2-DE were performed using proteins heated at 100°C for different periods of time, and 17 protein bands/spots were positively identified by MALDI TOF/TOF MS/MS. Protein profiles on both 1-DE and 2-DE gels changed obviously and more polydisperse bands/spots were observed with increased heating time for over-heated samples. Based on these observations, an alternative protein marker-producing method was designed by directly dissolving protein standards without BSA into lysis buffer. This new kind of protein marker could be stored at room temperature for a long time, thus was more convenient for using and shipping. The identification of 17 proteins via MS and comparison of their identities revealed MASCOT-searched scores, number of both matched peptides, total searched peptides and sequence coverage became progressively lower with increasing denaturation intensity, probably due to the interference of thermal denaturation on trypsin cleavage efficiency and produced redundant modified peptides. Therefore, it was concluded that thermal denaturation not only changed the protein profiles and produced more polydisperse protein bands/spots, but also heavily interfered with the subsequent MS analysis, hence not recommended in future proteomic analysis for proteins dissolved in lysis buffer.     

Explorative data analysis of two-dimensional electrophoresis gels

Methods for classification of two-dimensional (2-DE) electrophoresis gels based on multivariate data analysis are demonstrated. Two-dimensional gels of ten wheat varieties are analyzed and it is demonstrated how to classify the wheat varieties in two qualities and a method for initial screening of gels is presented. First, an approach is demonstrated in which no prior knowledge of the separated proteins is used. Alignment of the gels followed by a simple transformation of data makes it possible to analyze the gels in an automated explorative manner by principal component analysis, to determine if the gels should be further analyzed. A more detailed approach is done by analyzing spot volume lists by principal components analysis and partial least square regression. The use of spot volume data offers a mean to investigate the spot pattern and link the classified protein patterns to distinct spots on the gels for further investigation. The explorative approach in analysis of 2-D gels makes it possible, in a fast and convenient way, to screen many gels in order to determine the protein patterns that form clusters and could be selected for further examination.     

An explanation of the achromatic bands produced by peroxidase isozymes in polyacrylamide electrophoresis gels stained for malate dehydrogenase.

When plant tissue extracts are electrophoresed on polyacrylamide gels and the gels are stained for malate dehydrogenase by the standard NAD-dependent dehydrogenase reaction, terminating in the formation of reduced Nitroblue Tetrazolium (NBT), achromatic bands, in addition to the expected chromatic bands, are observed. The achromatic bands are seen when the staining conditions favor a generalized background staining of the gel and have been shown, in a previous study, to be caused by peroxidase isozymes [1]. The present study examined the mechanism by which peroxidase produced the achromatic bands using horseradish peroxidase (HRP). The generalized background staining resulted from the phenazine methosulfate (PMS)-mediated reduction of NBT. This reduction was enhanced by H2O2 and suppressed by HRP. Peroxidase apparently catalyzes the peroxidative oxidation of reduced PMS, which suppresses the generalized reduction of NBT in gel regions containing peroxidase isozymes producing the achromatic bands. In contrast, however, HRP also appears to catalyze the peroxidative oxidation of reduced NAD, but this reaction increases the reduction of NBT. The results are discussed in the context of the mechanisms proposed by others for the PMS-mediated reduction of NBT and for the peroxidase-catalyzed NADH-dependent formation of H2O2. This peroxidase-catalyzed reaction has been proposed for the plant peroxidases involved in lignification.     

CIEF with hydrodynamic and chemical mobilization for the separation of forms of alpha-1-acid glycoprotein

Alpha-1-acid glycoprotein (AGP) is a protein that exists in different forms, which is due to variations in the amino acid sequence and/or in the glycosidic part of the protein. These differences confer to these forms, among other characteristics, diverse pIs. Changes in these forms of AGP have been correlated to modifications of the pathophysiological conditions of the individuals. One of the analytical techniques employed for their study has been IEF performed in slab gels. CIEF method with hydrodynamic and chemical mobilization, involving an isotachophoretic process, is developed in this work to separate up to 12 bands of forms of standard AGP, which is proposed as a more reproducible, quantitative, less sample-consuming, and more automated one than conventional IEF. The challenge of this work has been the development of a CIEF method for the separation of bands of a very acidic protein (pI range: 1.8-3.8) in a capillary. Intraday RSD values < or = 1.7% have been achieved for the relative migration time of the AGP bands to that of an internal standard. For intraday area precision, RSD (%) in the range of 2.70-22.71% for AGP zones accounting for more than 10% of total area of AGP sample has been obtained. As a proof of the potential of the methodology proposed, an AGP sample purified from a pool of sera of patients suffering from ovary cancer is analyzed by CIEF.     

Multivariate analysis of 2-DE protein patterns--practical approaches

Practical approaches to the use of multivariate data analysis of 2-DE protein patterns are demonstrated by three independent strategies for the image analysis and the multivariate analysis on the same set of 2-DE data. Four wheat varieties were selected on the basis of their baking quality. Two of the varieties were of strong baking quality and hard wheat kernel and two were of weak baking quality and soft kernel. Gliadins at different stages of grain development were analyzed by the application of multivariate data analysis on images of 2-DEs. Patterns related to the wheat varieties, harvest times and quality were detected on images of 2-DE protein patterns for all the three strategies. The use of the multivariate methods was evaluated in the alignment and matching procedures of 2-DE gels. All the three strategies were able to discriminate the samples according to quality, harvest time and variety, although different subsets of protein spots were selected. The explorative approach of using multivariate data analysis and variable selection in the analyses of 2-DEs seems to be promising as a fast, reliable and convenient way of screening and transforming many gel images into spot quantities.     

Bis(amino acid) oxalyl amides as ambidextrous gelators of water and organic solvents: supramolecular gels with temperature dependent assembly/dissolution equilibrium

Bis(LeuOH) (1a), bis-(ValOH) (2a) and bis(PhgOH) (5a) (Phg denotes (R)-phenylglycine) oxalyl amides are efficient low molecular weight organic gelators of various organic solvents and their mixtures as well as water, water/DMSO, and water/DMF mixtures. The organisational motifs in aqueous gels are dominated primarily by lipophilic interactions while those in organic solvents are formed by intermolecular hydrogen bonding. Most of the gels are thermoreversible and stable for many months. However, 2a forms unstable gels with organic solvents which upon ageing transform into variety of crystalline shapes. For some 1a/alcohol gels, a linear correlation between alcohol dielectric constants (epsilon) and gel melting temperatures (Tg) was found. The 1H NMR and FTIR spectroscopic investigations of selected gels reveal the existence of temperature dependent network assembly/dissolution equilibrium. In the 1H NMR spectra of gels only the molecules dissolved in entrapped solvent could be observed. By using an internal standard, the concentration of dissolved gelator molecules could be determined. In FTIR spectra, the bands corresponding to network assembled and dissolved gelator molecules are simultaneously present. This enabled determination of the Kgel values by using both methods. From the plots of InKgel versus 1/T, the deltaHgel values of selected gels have been determined (-deltaHgel in 10-36 kJ mol(-1) range) and found to be strongly solvent dependent. The deltaHgel values determined by 1H NMR and FTIR spectroscopy are in excellent agreement. Crystal structures of 2a and rac-5a show the presence of organisational motifs and intermolecular interactions in agreement with those in gel fibres elucidated by spectroscopic methods.     

An improved methodology for adsorption characterization of unmodified and modified silica gels

An improvement in the adsorption characterization of the surface and structural properties of unmodified and modified mesoporous silica gels is presented. This improvement was achieved by selection of proper macroporous silica as the reference solid for adsorption characterization of porous silica gels. Experimental illustration is provided for unmodified and n-octyl-modified silica gels of different bonding density. The surface and structural properties of these silica gels were characterized by utilizing the standard adsorption data for both unmodified and octyl-modified LiChrospher Si-1000 macroporous silica gels. It was shown that the standard nitrogen adsorption data have an appreciable influence on the analysis of the pore size and surface properties of silica gels. This analysis can be improved by selecting the reference solid of the surface properties close to those of the silica gel studied.     

Recombinant autofluorescent landmarks for standardization of electrophoretic migration of proteins

Unequivocal identification of unknown protein spot patterns in two-dimensional (2-D) electrophoresis still represents a major problem when performing comparative studies of different 2-D electrophoresis gels. Inhomogeneity of gels due to variations in the gel casting procedure, electroendoosmosis and heterogeneity of proteins are major contributions to variations in migration patterns. By fusing green fluorescent protein to a number of well-defined selected proteins (human lysozyme, initiation factor 5a (EIF5a), rapamycin-selective 25 kDa immunophilin (FKBP25), and heat shock protein 90 beta (hsp90)), the isoelectric points and the molecular mass were designed. Proteins were additionally tagged with the FLAG tag enabling rapid purification by immunoaffinity chromatography. The fusion proteins were expressed intracellularly in yeast to avoid heterogeneity caused by post-translational modifications. The quality and applicability was tested in 1-D and 2-D electrophoresis. Sharp bands or symmetric spots were obtained. The proteins are considered as a new generation of reference proteins for electrokinetic separation methods.     

A procedure for analysis of densitometric spectra

Computer-aided quantitative analysis of densitometric spectra is presented. The densitometric spectra are decomposed into component bands using the Powell and Marquardt minimizers. Several different functions for the component bands are utilized. It is shown that the densitometric spectra can be decomposed into component bands with high accuracy only if the proper shapes of the bands are chosen. The method described was used for quantitative analysis of densitometric spectra of DNA cleaved by neocarzinostatin. The procedure is general and can be applied to analyses of autoradiographic films and to direct scans of electrophoretic gels. It is shown that densitometric spectra which show highly overlapped bands are well approximated by asymmetric Cauchy and Gauss functions. Well separated densitometric bands which have substantial asymmetry can be fitted to more sophisticated shapes formed by combinations of symmetric and asymmetric Cauchy and Gauss functions. High accuracy fitting to asymmetric densitometric curves may only be achieved using the cosine function introduced by Mignot and Rondot, J. Appl. Cryst. 1976, 9,460-465.     

Fluorescent labeling of proteins with nile red and 2-methoxy-2,4-diphenyl-3(2H)-furanone: physicochemical basis and application to the rapid staining of sodium dodecyl sulfate polyacrylamide gels and Western blots

The fluorescent hydrophobic dye Nile red allows the rapid, sensitive, and general staining of proteins in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Nile red staining does not preclude further electroblotting of protein bands onto polyvinylidene difluoride (PVDF) membranes. The resulting Western blot can be stained with the covalent fluorescent dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) using a simple procedure. MDPF staining allows further N-terminal microsequencing and immunodetection of specific bands. This review considers the physicochemical, structural, and analytical studies that have led to the development of Nile red and MDPF staining methods. The usefulness of these procedures is discussed in comparison to other currently available fluorescent and nonfluorescent protein detection methods.     

Use of a bilayer stacking gel to improve resolution of lipopolysaccharides and lipooligosaccharides in polyacrylamide gels

Lipopolysaccharide (LPS) and lipooligosaccharide (LOS) are important antigenic and integral components of the outer membrane of Gram-negative bacteria. Alteration or heterogeneity of LPS/LOS structure is most often assessed by alteration of electrophoretic band profiles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In order to discern minor differences in the electrophoretic profile of closely spaced bands, particularly the low molecular weight bands of LOS, optimum resolution is required. Unfortunately, many publications of LPS/LOS in polyacrylamide gels show a diffuse, smeared pattern without discernible bands. We report here a formulation for polyacrylamide gels that reproducibly yields LPS/LOS bands with sharp resolution. A key feature of this formulation is the use of a separate comb gel containing electrode buffer layered on top of the conventional stacking gel.     

General approach to low-molecular-weight metallogelators via the coordination-induced gelation of an L-glutamate-based lipid

A twin-tailed glutamate-based lipid with a pyridine headgroup was prepared in good yield using standard amide coupling and protection/deprotection chemistry. The resulting Lewis basic lipid gels a wide array of hydrocarbon solvents at a critical gelation concentration (C(g)) of 0.3 wt %. The gelation of more polar solvents, such as ethanol, THF, dichloromethane, and chloroform, occurs with a C(g) of between 2 and 5 wt %, demonstrating the importance of hydrogen bonding interactions in gel formation. The importance of hydrogen bonding in this system was also demonstrated by IR observation of the amide bands, which show a substantial shift upon gelation. Solutions of this new organogelator with concentrations below C(g) rapidly form gels upon the introduction of a wide variety of metal salts or complexes, providing a convenient general method for the preparation of metallogelators. Spectroscopic evidence suggests that the enhanced gelation seen in the metal-containing systems can be explained by a cross-linking of gel fibril aggregates similar to those formed by the unmetalated gelator.