Isolation and Identification of Specific Cortical Proteins in Tetrahymena pyriformis strain GL*†

SYNOPSIS. Pellicles of the ciliate Tetrahymena pyriformis strain GL (phenoset A) were isolated by a new procedure. Oral apparatuses were also purified by a modification of a previous method. Both preparations were characterized by electron microscopy. Proteins of the isolates were separated by analytical SDS polyacrylamide gel electrophoresis. The isolated pellicles, which included oral apparatuses, contained only 6 major proteins (gel bands), designated A through F. Bands A, B, and C, were found in the pellicle fraction, but not in the oral apparatus fraction. Therefore, these proteins are believed to be present in the somatic cortex of Tetrahymena. Bands D and E were greatly enriched in the oral apparatus fraction; these proteins are therefore believed to be present primarily in the oral apparatus. Band F, identified as tubulin, was present in both preparations. Molecular weight determinations and some selective solubilization experiments are also presented.     

Apparatus and method for preparative gel electrophoresis

A new apparatus for preparative gel electrophoresis with continuous elution which includes a miniaturized electrode and elution chamber system is described. The design provides high resolution, high yield, applicability for small and large amounts of peptide material, and easy operation. Furthermore, the apparatus enables a very accurate gel column or gel gradient to be formed. A method for preparative gel electrophoresis in sodium dodecyl sulfate which allows the purification of peptides and proteins without concurrently modifying tryptophane residues or blocking N-terminal α-amino groups is also described.     

A simple and accurate method for generating co-dominant markers: an application of conformation-sensitive gel electrophoresis to linkage analysis in the silkworm

A simple and sensitive method for linkage analysis is described, which is based on conformation-sensitive gel electrophoresis (CSGE). Using urea-containing agarose gels or a commercially available polyacrylamide-derived matrix, 13 polymorphic markers were newly identified for known genes of the silkworm, Bombyx mori, which had been scored as monomorphic by PCR-RFLP analysis. This method for detecting polymorphisms is quite sensitive, and can be performed with inexpensive reagents and apparatus that is available in most molecular biology laboratories. Received: 19 November 1998 / Accepted: 2 March 1999     

The isolation of high molecular weight DNA from wheat,barley and rye for analysis by pulse-field gel electrophoresis

A method is presented for the preparation of large DNA molecules from protoplasts embedded in agarose blocks of three different cereals-hexaploid bread wheat (Triticum aestivum), barley (Hordeum vulgare) and rye (Secale cereale). Pulse-field gel electrophoresis (PFGE) analysis of these DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA molecules was greater than 6 Mb. DNA samples prepared by this method were shown to be useful for restriction analysis using both frequent and rare cutting enzymes.     

Device for miniscale isoelectric focusing of proteins

A simple device is developed for mini-scale electrofocusing of proteins. The main apparatus consists of only two glass tubes joined by a small tubing. No special cooling system, stopcocks, stands, etc., are needed. Even the need for a peristaltic pump for fractionation is eliminated. The apparatus does not require very high voltages and the amount of Ampholines is drastically reduced. The model can be used for analytical as well as semi-quantitative purposes.     

An optimized protocol for the production of interdelta markers in Saccharomyces cerevisiae by using capillary electrophoresis

The amplification of genomic sequence blocks flanked by delta elements of retrotransposon origin has proved to be a very convenient method for molecular characterization of Saccharomyces cerevisiae strains. Fluorescent automated capillary electrophoresis (CE) was used to detect interdelta marker (IDM) patterns in S. cerevisiae, using the ABI Prism 3130 Genetic Analyzer. Main experimental parameters were studied and the optimal conditions for IDM amplification and samples run on the CE apparatus were determined. Fingerprints from fluorescent-labelled IDM produced using CE with the same sample analyzed by agarose electrophoresis (AE) were compared. The CE analysis was able to distinguish 43 different IDM profiles among 45 S. cerevisiae isolates with a discriminating capacity of 99.8%, whereas the AE analysis of the same samples allowed the identification of 27 different patterns (discriminatory power equal to 96%). Detection of fluorescent IDM was fast and reliable, and it facilitated data comparison. For the first time in our knowledge, the fluorescent CE proved to be well suited for IDM fingerprinting. Moreover, it could be routinely applied for the molecular differentiation of S. cerevisiae strains.     

Evaluation of isoelectric focusing running conditions during two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis: variation of gel patterns with changing conditions and optimized isoelectric focusing conditions

Five major isoelectric focusing (IEF) parameters--volt-hours; concentrations of acrylamide, NaOH, and H3PO4; and equilibration time--were systematically varied to determine the effect of each on two-dimensional IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel patterns and to optimize IEF conditions. Alterations in each parameter affected the gel pattern, frequently causing uncertainty in the identification of spots between conditions. The results emphasize the need for internal analytical consistency, and indicate that gel pattern comparisons between laboratories can be complicated if different IEF conditions are employed. The systematic evaluation indicated that optimized patterns were obtained when increased concentrations of NaOH and H3PO4 (to 50 and 25 mM, respectively) and run durations of 10,000 V-h or longer were used.     

Separation of biomacromolecules by electrofiltration through gel layers

A straightforward method for concomitant separation and isolation of biomacromolecules from a mixture in solution was developed. Three gel layers that comprise a middle separation layer of 10% polyacrylamide gel were constructed. This gel system was formed in an electroconcentration apparatus above a collection chamber surrounded at the bottom by a dialysis membrane. The mixture is applied over the gel layers where biomacromolecules are caused to migrate by electrophoresis through the gel system, where they are separated into discrete bands and electroeluted into the collection chamber without dismantling the apparatus. The isolated biomacromolecules are removed from the chamber in a highly pure and concentrated form ready for further investigations. Cooling can be applied throughout the whole process, and the setup and conditions of run can be modified according to the characteristics of the biomacromolecules to be purified. The components of a mixture containing the glycoprotein ovalbumin and bovine serum albumin monomer, dimer, and tetramer were successfully isolated as concentrated and highly pure fractions with good recoveries ranging from 70 to 89%. Other proteins were successfully isolated under denaturing conditions in the presence of sodium dodecyl sulfate (SDS) or 6 M urea.     

Gradient flattening of sodium dodecyl sulfate (SDS) and isoelectric focusing (IEF) gels

In addition to our previously reported versatile methods for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis [1] and isoelectric focusing [IEF]-gel [2], I have achieved molecular weight gradient flattening of the SDS-polyacrylamide gel and pH gradient flattening of the IEF gel at any segment using the same electrophoresis system. Any crowded gel segment where congregated components are not separated well can easily be widened for good separation and any dispersed gel segment where components are too far can easily be narrowed. Therefore, every gel segment can be used effectively and meaningfully because the gradient curve can be ajusted to any distribution of the components. In the crowded area, any small spots of components which could not be detected previously because of nearby heavy staining or strong radioactivity of an abundant component can be sufficiently separated from the nearby spots in a small gel without sacrificing other areas.     

Cost‐effective media for the rapid and high resolution of small DNA fragments using polyacrylamide‐based electrophoresis

Current Tris‐based solutions for DNA electrophoresis produce a positive feedback loop between current and temperature at high voltage, resulting in long running times for the separation of even small DNA fragments. We optimized the separation of small DNA fragments (90–300 bp) in polyacrylamide‐based electrophoresis at high voltages (200volts/cm) by substituting Tris with low concentration alkali salts (e.g. 1 mm LiCl and CsCl). These media reduced the heat produced during electrophoresis, enhanced the DNA fragment resolution, and allowed gels to be run at higher voltages, reducing gel running times by 25%. In addition, the elimination of Tris and EDTA from the buffer reduced material costs approximately 10‐fold.     

Analysis of ultra acidic proteins by the use of anodic acidic gels

Ultra acidic proteins, generated by posttranslational modifications, are becoming increasingly important due to recent evidence showing their function as regulatory elements or as intermediates in degradation pathways in bacteria. Such proteins are important in neurodegenerative diseases and embryonic development, and they include the Alzheimer-related tau (τ) protein (resulting from posttranslational modifications) and the phosphor-storage embryonic proteins. The ultra acidic proteins are difficult to study because standard two-dimensional gel electrophoresis is inadequate for their analysis. Here we describe a novel electrophoresis system of anodic acidic gels that can replace isoelectric focusing as the first dimension of separation in two-dimensional electrophoresis. The system is based on a sodium acetate buffer (pH 4.6), is compatible with traditional stains (e.g., Coomassie blue) as well as novel fluorescent dyes (e.g., Pro-Q Diamond), and is quantitative for the analysis of ultra acidic proteins. The anodic acidic gels were used for the functional classification of the ultra acidic part of the Bacillus subtilis proteome, showing significant improvement over traditional two-dimensional electrophoresis.     

Purification and Kinetic Characterisation of Lactate Dehydrogenase from Dioscorea cayenensis Tuber

Lactate dehydrogenase from yellow yam tuber (Dioscorea cayenensis Lam.) was isolated and purified using various chromatographic methods and electrophoresis. Only one form of the enzyme obtained, which obeyed Michaelis-Menten kinetics, was activated by Mg²⁺ and Ca²⁺ and inhibited by nucleotides and PEP. AMP, which activated the enzyme in the direction of pyruvate reduction, inhibited it in the direction of lactate oxidation. The enzyme is specific for pyruvate L-lactate and uses only NADH and NAD⁺ as the electron carriers. Polyacrylamide gel electrophoresis showed single band of lactate dehydrogenase activity. The average molecular mass obtained for the enzyme was 160 ± 1.2 kDa, while SDS gel electrophoresis indicated a dimer for the enzyme protein. The enzyme is very stable when frozen but its activity was hardly detectable when the tubers were stored in a well aerated place.     

Improved rapid detection of trypsin isoinhibitors using non-denaturing polyacrylamide gels with immobilised azoalbumin

Introduction – Proteinaceous inhibitors of animal trypsin occur naturally as isoforms in seeds and some are of interest as antinutritional or anti‐pest agents. Objective – To establish a simplified electrophorectic, in‐gel method for rapid and direct detection of trypsin isoinhibitors present in crude plant extracts that are particularly suitable for many studies including rapid evaluation of cultivars. Methodology – Azoalbumin (3%, w/v) is immobilised in 7.5% polyacrylamide gels before electrophoresis under non‐denaturing conditions. Results – This improved method eliminates the need for both time‐consuming and labourious staining and destaining or renaturation steps. Conclusion – Immobilised azoalbumin in polyacrylamide gels, run under non‐denaturing electrophoresis conditions, can be used to assist rapid evaluation of trypsin isoinhibitors in numerous crude plant extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparison of variability associated with sample preparation in two-dimensional gel electrophoresis of cardiac tissue.

Variability is a major complicating factor in analysis by two-dimensional gel electrophoresis. Improvements in methodologies have focused on improving individual gel quality rather than reproducibility. We homogenized rat cardiac tissue and rehydrated using a matrix of buffers to determine the optimal sample conditions. Six buffers were used to solubilize the proteins. Solubilized proteins were separated by isoelectric focusing using four buffers. Gels were run in triplicate to assess the method of preparation yielding the least variability. Number of spots and variability were different between conditions. Proteins solubilized in a buffer containing 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10, ampholytes, DTT, and protease inhibitors and focused in a buffer containing 9 M urea and 4% NP40 had the lowest coefficient of variation. Variability was compared across isoelectric point ranges and was different. Minimizing technical variability in two-dimensional polyacrylamide gel electrophoresis is critical to identify differences between conditions. Sample preparation should be optimized to minimize variability as well as to maximize the number of spots seen.     

Isoelectric focusing with reduced cathodic drift and migration into the anode chamber

An apparatus has been developed to reduce cathodic drift and migration into the anode chamber in vertical gel rod isoelectric focusing (IEF). In contrast to commercially available apparatuses, this apparatus can easily handle many more gels at one time, and the length, diameter and shape of its gel can be arbitrarily changed. In addition, high concentrations of detergent can be used to dissolve the protein samples, and removal of the gel cylinders from the glass tubes is easy.     

一步法电泳分析肌联蛋白亚型和肌球蛋白重链

目的为研究超大分子量肌小节蛋白肌联蛋白(titin)的生理病理功能,在一次电泳过程中同时分离titin各亚型和中分子量肌小节蛋白肌球蛋白重链(myosin heavy chain,MHC)。方法使用16cm×18cm垂直电泳系统,在电泳板下1/3灌注10g/L SDS-PAGE胶,上2/3灌注60g/L SDS-琼脂糖(SDS-VAGE)胶。低温8℃下持续电泳5h,在电泳板上层以SDS-VAGE胶电泳分离titin亚型,下层以SDS-PAGE胶电泳分离MHC。电泳后VAGE胶使用银染法标记titin各亚型,PAGE胶使用考马斯亮蓝染色法标记MHC。结果 titin各亚型得到有效的分离,目标蛋白条带显示清晰,与其分子量大小一一对应,分离效果明确。结论一步法垂直电泳系统可应用于超大分子量蛋白的电泳,同时可分离多个分子量差距大的蛋白,提高蛋白电泳实验效率。     

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method¹. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 °C during the gel run allows for the separation of unstructured DNA or RNA molecules.In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol ^1,2.     

Characterization of the filamentous hemagglutin from Bordetella pertussis by gel electrophoresis

Summary A highly purified preparation of filamentous hemagglutinin (FHA) from Bordetella pertussis was analyzed for its protein composition by gel electrophoretic methods. In this preparation of FHA the following native species could be detected by polyacrylamide gel electrophoresis (PAGE) at pH 3.2: S, and S₂ (inactive subunits or fragments); two monomers, a major form designated Ia (144K), and a minor form lb, differing only in net charge; and three oligomeric forms, designated II (213K), III (595K) and IV (1064K). Hemagglutinating activity was associated predominantly with component Ia. PAGE of FHA after derivatization with sodium dodecyl sulfate (SDS) showed there to be three major species, designated A, C and D. According to estimated molecular weight values, A, C and D are likely to correspond to S₂, Ia and II respectively. Isolated components II, III and IV yield all three SDS-species upon derivatization with SDS. Both moving boundary electrophoresis and gel electrofocusing showed hemagglutinating FHA to be a basic protein. Its apparent pI is 8.1.     

Comparative proteome analysis of breast cancer and normal breast

Breast cancer is a leading cause of death for women. The underlying molecular mechanism is still not well understood. In this study, two-dimensional gel electrophoresis combined with mass spectrometry was used to analyze changes in the proteome of infiltrating ductal carcinoma compared to normal breast tissue. Ten sets of two-dimensional gels per experimental condition were analyzed and more than 500 spots each were detected. This revealed 39 spots for which expression in breast cancer cells were reproducibly altered more than twofold compared to normal controls (p<0.01). These spots represented 25 different proteins after identification using the database search after mass spectrometry, comprising cell defense proteins, enzymes involved in glycolytic energy metabolism and homeostasis, protein folding and structural proteins, proteins involved in cytoskeleton and cell motility, and proteins involved in other functions. In addition, 28 nondifferentially expressed proteins with different functions were also mapped and identified, which might help to establish a two-dimensional gel electrophoresis reference map of human breast cancer. Our study shows that proteomics offers a powerful methodology to detect the proteins that show different expression patterns in breast cancer tissue and may provide an accurate molecular classification. The differentially expressed proteins may be used as potential candidate markers for diagnostic purposes or for determination of tumor sensitivity to therapy. The functional implications of the identified proteins are discussed.     

A vertical submarine electrophoresis apparatus for polyacrylamide minigels

A vertical submarine electrophoresis apparatus for use with minislab polyacrylamide gels is described. The design allows polyacrylamide gels to be run with the same ease and convenience that agarose gels are run with horizontal submarine apparatuses. The vertical submarine features a single buffer chamber with a restriction between the upper and the lower portions of the chamber. Acrylamide gels, cast between 9 X 10-cm glass slides, are inserted into the restriction and are completely immersed in buffer. Thus, current flows primarily through the gel itself, but some current flows through the buffer in the restriction surrounding the gel. Because water-tight separation of buffer chambers is not necessary, time-consuming and/or expensive procedures such as sealing with agarose or using fragile notched glass plates are eliminated. The apparatus can be set up to run a gel in less than 30 s. It is versatile in that gels of varying thickness (0.5, 0.8, 1.5, and 3 mm) can be run on a single apparatus. The apparatus has been used for sodium dodecyl sulfate gels, low ionic strength native gels for nucleoprotein complexes, and composite acrylamide-agarose gels.