Analysis of Protein Glycation Using Phenylboronate Acrylamide Gel Electrophoresis.

Carbohydrate modification of proteins adds complexity and diversity to the proteome. However, undesired carbohydrate modifications also occur in the form of glycation, which have been implicated in diseases such as diabetes, Alzheimer's disease, autoimmune diseases, and cancer. The analysis of glycated proteins is challenging due to their complexity and variability. Numerous analytical techniques have been developed that require expensive specialized equipment and complex data analysis. In this chapter, we describe two easy-to-use electrophoresis-based methods that will enable researchers to detect, identify, and analyze these posttranslational modifications. This new cost-effective methodology will aid the detection of unwanted glycation products in processed foods and may lead to new diagnostics and therapeutics for age-related chronic diseases.

Polyacrylamide gel photopatterning enables automated protein immunoblotting in a two-dimensional microdevice.

We demonstrate a two-dimensional microfluidic architecture that integrates polyacrylamide gel electrophoresis (PAGE) with immunoblotting in a fully automated format. This assay is designed to overcome performance limitations of conventional slab-gel immunoblotting, including multiple manual interventions, low-throughput transfer and blotting, and substantial consumption of reagents and sample. To unify PAGE with blotting in one device, this microfluidic approach makes use of high-resolution regional photopatterning of multiple polyacrylamide gel elements, and automated electrophoretic transport. A complete native immunoblot of free prostate specific antigen from human seminal fluid is demonstrated in less than 5 min. Further, the characterization of post-PAGE electrophoretic transfer showed high efficiency and minimal sample dispersion.

Determination of DNA methylation by COBRA: a comparative study of CGE with LIF detection and conventional gel electrophoresis.

DNA methylation as an epigenetic modification of the human genome is under emphatic investigation. Several studies have demonstrated a role of DNA methylation in oncogenesis. In conjunction with histone modifications, DNA methylation may cause the formation of heterochromatin and thus mediate the inactivation of gene transcription. It is important to develop methods that allow for an accurate quantification of the amount of DNA methylation in particular DNA regions, to gain information concerning the threshold of methylation levels necessary for gene inactivation. In this article, a CGE method with on-column LIF detection using SYBR Green is compared with a conventional slab-gel electrophoresis. We thus investigate the validity to analyze DNA methylation in the samples of a combined bisulfite restriction analysis. It is demonstrated that CGE is superior to gel electrophoresis in means of linearity, precision, accuracy, automatization (high throughput), and sample consumption. However, gel electrophoresis is easier to perform (simple devices, no PC usage), and the running costs are comparatively low. A further advantage of CGE is the sparse use of toxic compounds (MeOH and SYBR Green), whereas gel electrophoresis is performed in polyacrylamide gels with ethidium bromide staining.

Electrophoresis: the march of pennies, the march of dimes.

The present review encompasses ca. 65 years of history of developments in electrokinetic separations, taking as a starting point the year 1937, i.e. the official launching of Tiselius' moving boundary electrophoresis (MBE). The 1950s have been particularly rich in introducing novel methodologies in zone electrophoresis (ZE), thus bringing about the decline of MBE. Among them of extraordinary importance was the development of electrophoresis on agar gels coupled to immuno-diffusion at right angles, which brought a big revolution not only in biochemistry but also in clinical chemistry. Also the by now forgotten paper electrophoresis was a landmark in separation science, in that it implemented, in its "fingerprinting" version, the first genuine two-dimensional (2D) map, coupling orthogonally a charge to a hydrophobic scale separation, while permitting for the first time the detection of spot mutations, i.e. single amino acid replacements in a polypeptide chain, that paved the way to modern genetic analysis. Equally important was the introduction of starch-block electrophoresis, that brought about the notion of sieving and the first discontinuous buffers, refined, in the 1960s, by Ornstein and Davies with their classical papers combining multiphasic buffer systems to polyacrylamide gels, that went down to history as disc-electrophoresis. The 1960s also contributed with two fundamental techniques, isoelectric focusing (IEF) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) that permitted to discriminate proteins solely on the basis of surface charge and molecular mass, respectively. The 1970s gave other fundamental contributions, such as isotachophoresis, the first example of a fully instrumental approach to electrophoresis, both in its analytical and preparative version (Tachophor and Tachofrac), 2D maps combining IEF to SDS-PAGE at right angles and silver staining techniques, that incremented sensitivity by 3 orders of magnitude. The 1980s generated immobilized pH gradients and capillary zone electrophoresis (CZE), two big players that dominated the electrokinetic horizon for all the 1990s and still in vigorous use in present days. The review terminates with a glimpse, in the third millennium, onto microchip technology and hyphenated techniques, notably direct interfacing of various electrophoretic separation methods with mass spectrometry (MS).

SDS-PAGE is underutilized as a tool for investigating renal patients.

SDS-PAGE is an excellent single test for investigating proteinuria. It can provide much useful information on the underlying renal problem. Yet the literature hardly report a SDS-PAGE result in the management of renal patients. To examine how closely SDS-PAGE results may reflect biopsy findings, we investigated 11 patients scheduled for renal biopsy. Urine samples were taken at the same time for SDS-PAGE analysis using the PhastSystem (Pharmacia, Sweden). Comparing biopsy findings and SDS-PAGE results, the data show consistency in the revelation of tubular dysfunction and/or glomerular damage in all 11 patients. We concluded that the SDS-PAGE test is underutilized and suggest that its role for the management of renal patients be fully explored particularly in its potential for reducing the need for renal biopsy in certain patient groups.

Detection of mutations in multi-exon genes: comparison of conformation sensitive gel electrophoresis and sequencing strategies with respect to cost and time for finding mutations.

This report compares the relative advantages and disadvantages of two alternative strategies with respect to cost and time for finding mutations in the COL2A1 gene in patients with degenerative diseases of the joint. The coding region of the COL2A1 gene, 30 kb in length and containing 52 exons, can be analyzed using 26 genomic PCR products. The results indicate that the most efficient and cost effective way to screen large genes is a prescreen of the coding sequences followed by sequencing of a limited number of variant-PCR products.

Rapid diagnosis of chronic myeloid leukaemia by linking PCR to capillary gel electrophoresis.

The move to the use of molecular diagnostics in medicine has gathered momentum in the last few years and new methodologies are being sought to reduce the labour component and hence the cost of these molecular diagnostics. A method is described which links PCR to capillary electrophoresis and this allows both a rapid and quantitative diagnosis. In the example provided, the diagnosis of chronic myeloid leukaemia, the method describes improvements in cost per test, greater sensitivity over traditional methods and an estimate of the level of product. The latter points are important in those disorders for which bone marrow transplantation is used as a curative regime in providing earlier detection of relapse.

Detection of exo-beta-1,3-glucanase activity in polyacrylamide gels after electrophoresis under denaturing or nondenaturing conditions.

A method for the visualization of exo-beta-1,3-glucanase activity in polyacrylamide gels is presented. The procedure consists of the enzyme reaction in the gel with the substrate alpha-naphthylglucopyranoside, and a subsequent staining of the obtained alpha-naphthol with dyes Fast Red B, or Fast Blue BB, respectively. A mixture of exoglucanases produced by the fungus Polyporus squamosus was used for the optimization of the method. The procedure is applicable for the standard Laemmli discontinuous electrophoresis system, even in the presence of sodium dodecyl sulfate, as well as for electrophoresis in linear gradients of the polyacrylamide concentration. The staining method was used for the analysis of exoglucanases secreted by several yeast genera. All yeasts tested produced two types of exoglucanases, a high molecular mass species heterogeneous in size, and one or two smaller homogeneous enzymes.

Eluiçäo de proteínas de gel de poliacrilamida: descriçäo de metodologia simples e econômica / Elution of proteins from polyacrylamide gel: a simple and economic procedure description

Descreve-se uma metodologia simplificada para a eletroeluiçäo quantitativa de proteína de gel de poliacrilamida. Após coloraçäo do gel pelo Coomassie Billiant Blue R 250, componentes identificados säo recortados e as proteínas eluidas do gel por um procedimento desenvolvido para uso em aparelho de eletroforese vertical em tubos