Showing papers on "Gel electrophoresis published in 2011"

Phosphorylation of Smooth Muscle Myosin Light Chain Kinase by the Catalytic Subunit of Adenosine 3': 5'-Monophosphate-dependent

Abstract: SUMMARY Turkey gizzard smooth muscle light chain kinase was purified by affinity chromatography on calcium dependent regulator coupled to Sepharose and found to have a molecular weight of 125,000 + 5,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When myosin light chain kinase is incubated with the catalytic subunit of cyclic AMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of myosin kinase. Brief tryptic digestion of the 32P-labeled myosin kinase liberates a single radioactive peptide with a molecular weight of approximately 22,000. Phosphorylation of myosin kinase results in a a-fold decrease in the rate at which the enzyme phosphorylates the 20,000-dalton light chain of smooth muscle myosin. These results suggest that cyclic AMP has a direct effect on actin-myosin interaction in smooth muscle.

Defining the Protein Complex Proteome of Plant Mitochondria

Abstract: A classical approach, protein separation by two-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was combined with tandem mass spectrometry and up-to-date computer technology to characterize the mitochondrial “protein complex proteome” of Arabidopsis (Arabidopsis thaliana) in so far unrivaled depth. We further developed the novel GelMap software package to annotate and evaluate two-dimensional blue native/sodium dodecyl sulfate gels. The software allows (1) annotation of proteins according to functional and structural correlations (e.g. subunits of a distinct protein complex), (2) assignment of comprehensive protein identification lists to individual gel spots, and thereby (3) selective display of protein complexes of low abundance. In total, 471 distinct proteins were identified by mass spectrometry, several of which form part of at least 35 different mitochondrial protein complexes. To our knowledge, numerous protein complexes were described for the first time (e.g. complexes including pentatricopeptide repeat proteins involved in nucleic acid metabolism). Discovery of further protein complexes within our data set is open to everybody via the public GelMap portal at www.gelmap.de/arabidopsis_mito.

High-abundance proteins depletion for serum proteomic analysis: concomitant removal of non-targeted proteins

Abstract: In clinical and pharmaceutical proteomics, serum and plasma are frequently used for detection of early diagnostic biomarkers for therapeutic targets. Although obtaining these body fluid samples is non-invasive and easy, they contain some abundant proteins that mask other protein components present at low concentrations. The challenge in identifying serum biomarkers is to remove the abundant proteins, uncovering and enriching at the same time the low-abundance ones. The depletion strategies, however, could lead to the concomitant removal of some non-targeted proteins that may be of potential interest. In this study, we compared three different methods aimed to deplete high-abundance proteins from human serum, focusing on the identification of non-specifically bound proteins which might be eventually removed. A Cibacron blue-dye-based method for albumin removal, an albumin and IgG immunodepletion method and an immunoaffinity column (Multiple Affinity Removal System) that simultaneously removes a total of six high-abundance proteins, were investigated. The bound proteins were eluted, separated by two-dimensional gel electrophoresis and identified by Nano LC-CHIP-MS system. Flow-through fractions and bound fractions were also analysed with the ProteinChip technology SELDI-TOF-MS. Our results showed that the methods tested removed not only the targeted proteins with high efficiency, but also some non-targeted proteins. We found that the Multiple Affinity Removal Column improved the intensity of low-abundance proteins, displayed new protein spots and increased resolution. Notably, the column showed the lowest removal of untargeted proteins, proved to be the most promising depletion approach and a reliable method for serum preparation prior to proteomic studies.

Reactivity of Human Salivary Proteins Families Toward Food Polyphenols

Abstract: Tannins are well-known food polyphenols that interact with proteins, namely, salivary proteins. This interaction is an important factor in relation to their bioavailability and is considered the basis of several important properties of tannins, namely, the development of astringency. It has been generally accepted that astringency is due to the tannin-induced complexation and/or precipitation of salivary proline-rich proteins (PRPs) in the oral cavity. However, this complexation is thought to provide protection against dietary tannins. Neverthless, there is no concrete evidence and agreement about which PRP families (acidic, basic, and glycosylated) are responsible for the interaction with condensed tannins. In the present work, human saliva was isolated, and the proteins existing in saliva were characterized by chromatographic and proteomic approaches (HPLC-DAD, ESI-MS, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and MALDI-TOF). These approaches were also adapted to study the affinity of the different families of salivary proteins to condensed tannins by the interaction of saliva with grape seed procyanidins. The results obtained when all the main families of salivary proteins are present in a competitive assay, like in the oral cavity, demonstrate that condensed tannins interact first with acidic PRPs and statherin and thereafter with histatins, glycosylated PRPs, and bPRPs.

Factors Influencing the DNA Nuclease Activity of Iron, Cobalt, Nickel, and Copper Chelates

Abstract: A library of complexes that included iron, cobalt, nickel, and copper chelates of cyclam, cyclen, DOTA, DTPA, EDTA, tripeptide GGH, tetrapeptide KGHK, NTA, and TACN was evaluated for DNA nuclease activity, ascorbate consumption, superoxide and hydroxyl radical generation, and reduction potential under physiologically relevant conditions. Plasmid DNA cleavage rates demonstrated by combinations of each complex and biological co-reactants were quantified by gel electrophoresis, yielding second-order rate constants for DNA(supercoiled) to DNA(nicked) conversion up to 2.5 × 10(6) M(-1) min(-1), and for DNA(nicked) to DNA(linear) up to 7 × 10(5) M(-1) min(-1). Relative rates of radical generation and characterization of radical species were determined by reaction with the fluorescent radical probes TEMPO-9-AC and rhodamine B. Ascorbate turnover rate constants ranging from 3 × 10(-4) to 0.13 min(-1) were determined, although many complexes demonstrated no measurable activity. Inhibition and Freifelder-Trumbo analysis of DNA cleavage supported concerted cleavage of dsDNA by a metal-associated reactive oxygen species (ROS) in the case of Cu(2+)(aq), Cu-KGHK, Co-KGHK, and Cu-NTA and stepwise cleavage for Fe(2+)(aq), Cu-cyclam, Cu-cyclen, Co-cyclen, Cu-EDTA, Ni-EDTA, Co-EDTA, Cu-GGH, and Co-NTA. Reduction potentials varied over the range from -362 to +1111 mV versus NHE, and complexes demonstrated optimal catalytic activity in the range of the physiological redox co-reactants ascorbate and peroxide (-66 to +380 mV).

Electrophoretic methods for the analysis of nanoparticles

Abstract: With the development of nanotechnology, there is a need for methodologies to determine and characterize nanomaterials. Electrophoresis has emerged as a useful tool, which has been employed in various formats (e.g., capillary-zone electrophoresis, gel electrophoresis or isotachophoresis) for the size- or shape-based separation of different types of nanoparticle (NP) (e.g., metallic, semi-metallic or carbon). This article reviews the main progress in electrophoresis techniques in order to achieve separation of NPs.

Lipoic acid modified low molecular weight polyethylenimine mediates nontoxic and highly potent in vitro gene transfection.

Abstract: The clinical success of gene therapy intimately relies on the development of safe and efficient gene carrier systems. We found here that 1.8 kDa polyethylenimine (PEI) following hydrophobic modification with lipoic acid (LA) mediated nontoxic and highly potent in vitro gene transfection in both HeLa and 293T cells. 1.8 kDa PEI–LA conjugates were prepared with controlled degree of substitution (DS) by coupling LA to PEI using carbodiimide chemistry. Gel electrophoresis measurements showed that the DNA binding ability of 1.8 kDa PEI was impaired by lipoylation, in which an N/P ratio of 2/1 and 4–6/1 was required for 1.8 kDa PEI and 1.8 kDa PEI–LA conjugates, respectively, to completely inhibit DNA migration. Interestingly, dynamic light scattering measurements (DLS) revealed that PEI–LA conjugates condensed DNA into much smaller sizes (183–84 nm) than unmodified 1.8 kDa PEI (444–139 nm) at N/P ratios ranging from 20/1 to 60/1. These polyplexes revealed similar surface charges of ca. +22 to +30 mV. 1.8 kDa P...

Purification and identification of sperm surface proteins and changes during epididymal maturation.

Abstract: Surface membrane proteins have a key role in the sequential interactions between spermatozoa and oocytes. The aim of this study was to characterize protein changes occurring during post-testicular differentiation using a new overall approach to study surface membrane proteins of spermatozoa. A dedicated protocol based on specific purification of surface membrane proteins labeled with sulfo-NHS-SS-biotin was developed for this purpose. Appropriate gel electrophoresis separation and purification methods combined with standard proteomic methods were then used to identify and quantify surface membrane proteins from immature and mature spermatozoa. Membrane-associated proteins were discriminated from integral membrane proteins by differential solubilization. Protein regionalization on the spermatozoon surface was achieved by comparative analysis of the surface protein extracts from the entire spermatozoa and from periacrosomal sperm plasma membranes. Identification of several known proteins and of new proteins related to the process of epididymal maturation showed the reliability of this protocol for specific purification of a subproteome and identification of new sperm membrane proteins. This approach opens up a new area in the search for male fertility markers.

Changes in tear protein profile in keratoconus disease.

Abstract: To analyze tear protein profile variations in patients with keratoconus (KC) and to compare them with those of control subjects. Tears from 12 normal subjects and 12 patients with KC were analyzed by two-dimensional gel electrophoresis (2-DE) and liquid chromatography–mass spectrometry (LC–MS). Analysis of the 2-DE gels was performed using Progenesis SameSpots software (Nonlinear Dynamics). Proteins exhibiting high variation in expression levels (P-value <0.05) were identified using matrix-assisted laser desorption/ionization–TOF spectrometry. For LC–MS analysis, a label-free quantification approach was used. Tears were digested with trypsin, subjected to data-independent acquisition (MSE) analysis, and identified proteins were relatively quantified using ProteinLynx Global Server software (Waters). The 2-DE and LC–MS analyses revealed a significant decrease in the levels of members of the cystatin family and an increase in lipocalin-1 in KC patients. A 1.43-fold decrease was observed for cystatin-S by 2-DE, and 1.69- and 1.56-fold for cystatin-SN and cystatin-SA by LC–MS, respectively. The increase in lipocalin-1 was observed by both methods with fold changes of 1.26 in the 2-DE approach and 1.31 according to LC–MS. Significant protein upregulation was also observed for Ig-κchain C and Ig J chain proteins by 2-DE. Levels of lipophilin-C, lipophilin-A, and phospholipase A2 were decreased in tears from KC patients according to LC–MS. Serum albumin was found to be increased in KC patients according to LC–MS. The results show differences in the tear protein profile of KC and control subjects. These changes are indicative of alterations in tear film stability and in interactions with the corneal surface in KC patients.

Proteomic analysis of seed germination under salt stress in soybeans

Abstract: Soybean (Glycine max (L.) Merrill) is a salt-sensitive crop, and its production is severely affected by saline soils. Therefore, the response of soybean seeds to salt stress during germination was investigated at both physiological and proteomic levels. The salt-tolerant cultivar Lee68 and salt-sensitive cultivar N2899 were exposed to 100 mmol/L NaCl until radicle protrusion from the seed coat. In both cultivars, the final germination percentage was not affected by salt, but the mean germination times of Lee68 and N2899 were delayed by 0.3 and 1.0 d, respectively, compared with controls. In response to salt stress, the abscisic acid content increased, and gibberellic acid (GA1+3) and isopentenyladenosine decreased. Indole-3-acetic acid increased in Lee68, but remained unchanged in N2899. The proteins extracted from germinated seeds were separated using two-dimensional gel electrophoresis (2-DE), followed by Coomassie brilliant blue G-250 staining. About 350 protein spots from 2-DE gels of pH range 3 to 10 and 650 spots from gels of pH range 4 to 7 were reproducibly resolved, of which 18 protein spots showed changes in abundance as a result of salt stress in both cultivars. After matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) analysis of the differentially expressed proteins, the peptide mass fingerprint was searched against the soybean UniGene database and nine proteins were successfully identified. Ferritin and 20S proteasome subunit β-6 were up-regulated in both cultivars. Glyceraldehyde 3-phosphate dehydrogenase, glutathione S-transferase (GST) 9, GST 10, and seed maturation protein PM36 were down-regulated in Lee68 by salt, but still remained at a certain level. However, these proteins were present in lower levels in control N2899 and were up-regulated under salt stress. The results indicate that these proteins might have important roles in defense mechanisms against salt stress during soybean seed germination.

Differences in DNA condensation and release by lysine and arginine homopeptides govern their DNA delivery efficiencies.

Abstract: Designing of nanocarriers that can efficiently deliver therapeutic DNA payload and allow its smooth intracellular release for transgene expression is still a major constraint. The optimization of DNA nanocarriers requires thorough understanding of the chemical and structural characteristics of the vector–nucleic acid complexes and its correlation with the cellular entry, intracellular state and transfection efficiency. l-Lysine and l-arginine based cationic peptides alone or in conjugation with other vectors are known to be putative DNA delivery agents. Here we have used l-lysine and l-arginine homopeptides of three different lengths and probed their DNA condensation and release properties by using a multitude of biophysical techniques including fluorescence spectroscopy, gel electrophoresis and atomic force microscopy. Our results clearly showed that although both lysine and arginine based homopeptides condense DNA via electrostatic interactions, they follow different pattern of DNA condensation and rele...

The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments

Abstract: The single cell gel electrophoresis (comet assay) is a popular method for measuring DNA migration as an estimate of DNA damage. No standardised comet assay protocol exists, which make comparisons between studies complicated. In a previous inter-laboratory validation study of the comet assay, we identified important parameters in the protocol that might affect DNA migration. The aim of this study was to assess how different comet assay protocols affect DNA migration. The results in this study suggest that (i) there is a significant linear dose-response relationship between the agarose gel's density and DNA migration and that damaged cells are more sensitive to the agarose gel's density; (ii) incubation with formamidopyrimidine DNA glycosylase for 10 min is inadequate, whereas 30 min is sufficient; (iii) the typically used 20 min of alkaline treatment might be to short when analysing samples that contain particular alkali-labile sites (ALS) and (iv) the duration of electrophoresis as well as the strength of the electric field applied affects the DNA migration. By using protocol-specific calibration curves, it is possible to reduce the variation in DNA migration caused by differences in comet assay protocols. This does, however, not completely remove the impact of the durations of alkaline treatment and electrophoresis when analysing cells containing ALS that are relatively resistant to high alkaline treatment.

A robust universal method for extraction of genomicdna from bacterial species

Abstract: Background and Objectives: The intactness of DNA is the keystone of genome-based clinical investigations, where rapid molecular detection of life-threatening bacteria is largely dependent on the isolation of high-quality DNA. Various protocols have been so far developed for genomic DNA isolation from bacteria, most of which have been claimed to be reproducible with relatively good yields of high-quality DNA. Nonetheless, they are not fully applicable to various types of bacteria, their processing cost is relatively high, and some toxic reagents are used. The routine protocols for DNA extraction appear to be sensitive to species diversity, and may fail to produce high-quality DNA from different species. Such protocols remain time-consuming and tedious, thus to resolve some of these impediments, we report development of a very simple, rapid, and high throughput protocol for extracting of high-quality DNA from different bacterial species. Methods: In so doing, 9 different bacterial species were selected based on the structural complexity of cell wall, extracellular material, economical importance and significance in clinical and biotechnological researches. In order to evaluate the quantity, quality, purity and intactness of the extracted DNA, gel electrophoresis, PCR, RAPD and restriction enzyme digestion methods were performed. Results: Based upon our protocol, interfering phenolic compounds were removed from extraction using poly vinyl pyrrolidone (PVP) and RNA contamination was precipitated using LiCl. The UV spectrophotometric and gel electrophoresis analysis resulted in high A260/A280 ratio (>1.8) with high intactness of DNA. Quality assessment with restriction digestion showed that the isolated DNA was very pure with high quality for enzymatic reaction, in which a minimal inhibitory effect of the extraction process was observed. The PCR product yielded a clear band pattern and adequate intensity. Conclusion: The isolated DNA from samples confirmed the accuracy of this protocol which requires no enzymatic processing and accordingly its low-cost making it an appropriate method for large-scale DNA isolation from various bacterial species in medical researches. This protocol could serve as a universal competent method for isolation of genomic DNA from a variety of microorganisms with different types of extracellular and dissimilar cellular envelops architecture.

Novel royal jelly proteins identified by gel-based and gel-free proteomics.

Abstract: Royal jelly (RJ) plays an important role in caste determination of the honeybee; the genetically same female egg develops into either a queen or worker bee depending on the time and amount of RJ fed to the larvae. RJ also has numerous health-promoting properties for humans. Gel-based and gel-free proteomics approaches and high-performance liquid chromatography–chip quadruple time-of-flight tandem mass spectrometry were applied to comprehensively investigate the protein components of RJ. Overall, 37 and 22 nonredundant proteins were identified by one-dimensional gel electrophoresis and gel-free analysis, respectively, and 19 new proteins were found by these two proteomics approaches. Major royal jelly proteins (MRJPs) were identified as the principal protein components of RJ, and proteins related to carbohydrate metabolism such as glucose oxidase, α-glucosidase precursor, and glucose dehydrogenase were also successfully identified. Importantly, the 19 newly identified proteins were mainly classified into t...

Western blotting using capillary electrophoresis.

Abstract: A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein adsorption. Although discrete protein zones could be detected, bands were broadened by ∼1.7-fold by transfer to membrane. A complete Western blot for lysozyme was completed in about one hour with 50 pg mass detection limit from low microgram per milliliter samples. These results demonstrate substantial reduction in time requirements and improvement in mass sensitivity compared to conventional Western blots. Western blotting using capillary electrophoresis shows promise to analyze low volume samples with reduced reagents and time, while retaining the information content of a typical Western blot.

A comparison of depletion versus equalization for reducing high-abundance proteins in human serum.

Abstract: In this work three methods to diminish the content of most highly abundant proteins in human serum have been studied and compared Protein depletion with ACN or DTT and protein equalization with the ProteoMiner(™) (PM) have been assessed by 1-D gel electrophoresis and MS After treatment 5, 18 and 9 major proteins within the 20 most abundant proteins in serum were identified for the ACN, DTT and PM methods, respectively The ACN method was efficient for depleting high molecular weight proteins, over 75 KDa, resulting in 10±4% (n=3) of the total protein content remaining in the depleted serum In addition, 75% of the proteins belonging to the group of the 20 most abundant proteins were not detected, making this depletion strategy a cheap alternative to expensive commercial tools regularly used for removing high abundance proteins from serum The ACN extract was found rich in apolipoproteins The dithithreitol method promotes the precipitation of proteins rich in disulfide bonds, mainly albumin, with 73±7% (n=3) of the total protein content remaining in the depleted serum, which was found rich in immunoglobulins The PM method compresses the dynamic range of the serum proteins, rendering an extract containing 16±2% (n=3) of the total initial protein content The extract was found to be rich in both apolipoproteins and immunoglobulins As a general rule the DTT and PM methods provide a compression of the dynamic range of serum protein concentrations while the ACN method allows an effective depletion of the protein fraction above 72 KDa

DNA-binding, oxidative DNA cleavage, and coordination mode of later 3d transition metal complexes of a Schiff base derived from isatin as antimicrobial agents

Abstract: A Schiff base, obtained by the condensation of isatin monohydrazone with 2,3,5-trichlorobenzaldehyde, and its Co(II), Ni(II), Cu(II), and Zn(II) complexes have been synthesized and characterized. The interaction of these complexes with DNA is investigated using viscosity, absorption titration, and electrochemical techniques. The results indicate that the complexes bind to Calf thymus DNA through intercalation. Oxidative cleavage activities of the complexes are studied using supercoiled pBR322 DNA by gel electrophoresis. Antimicrobial study reveals that copper and zinc complexes are better antimicrobial agents than the Schiff base and its other complexes.

Gel-Based and Gel-Free Proteomic Technologies

Abstract: Proteomics refers to the analysis of expression, localization, functions, posttranslational modifications, and interactions of proteins expressed by a genome at a specific condition and at a specific time. Mass spectrometry (MS)-based proteomic methods have emerged as a key technology for unbiased systematic and high-throughput identification and quantification of complex protein mixtures. These methods have the potential to reveal unknown and novel changes in protein interactions and assemblies that regulate cellular and physiological processes. Both gel-based (one-dimensional [1D] gel electrophoresis, two-dimensional [2D] polyacrylamide gel electrophoresis, 2D difference in-gel electrophoresis [DIGE]) and gel-free (liquid chromatography [LC], capillary electrophoresis) approaches have been developed and utilized in a variety of combinations to separate proteins prior to mass spectrometric analysis. Detailed protocols for global proteomic analysis from adipose-derived stem cells (ASCs) using two central strategies, 2D-DIGE-MS and 2D-LC-MS, are presented here.