Dimensional Electrophoresis Gels Research Articles

Protein Detection of Excretory-Secretory Products and Somatic Extracts from Fasciola hepatica and F. gigantica Using Two-Dimensional Electrophoresis

The aim of this research was to compare excretory-secretory and somatic extract materials of Fasciola hepatica and F. gigantica to detect protein maps of two species. Twenty infected livers were collected from sheep in industrial slaughterhouse in Tehran, 2017-2019. Worms were detached from bile ducts, then recognized according to morphologic and morphometric criteria. After three times washing, worms were incubated in RPMI culture media and excretory-secretory products were collected. Worms were crushed and homogenized for preparation of somatic extract. Two Dimensional Electrophoresis gels were accomplished for both excretory-secretory material and somatic extracts. Gels were scanned with densitometer and analyzed with Same Spots software and protein spots were identified with Expasy database. For both excretory-secretory products and somatic extract, protein spots were appeared with two-dimensional electrophoresis technique. Quantitative analysis showed 40 and 28 protein spots for excretory-secretory of F. hepatica and F. gigantica respectively. For somatic extract 19 and 12 protein spots were recognized for F. hepatica and F. gigantica in that order. The rate of expression of some proteins were more in F. hepatica while expression of other proteins was high in F. gigantica. The expression of protease enzyme was higher in F. gigantica than F. hepatica. These data could be considered for biochemical differentiation of Fasciola species and subsequently to design and prepare of antigens for diagnosis/vaccine development.

Proteomics Analysis of Photo-Thermo-Sensitive Male Sterility Wheat Line BNS during Its Thermosensitive Period

Photo-thermo sensitive male sterile (PTMS) line is one of the important materials in utilizing heterosis in crops. Wheat line BNS (Bainong sterility) is an important nuclear-controlling PTMS line and suitable for growing and seed production in Huang Huai wheat zone in China. It has genetic stability with male sterility when sowing in autumn and male fertility when sowing in spring. Their thermosensitive periods were between stamen and pistil differentiation stage and anther connective stage and they could be regarded key periods for fertility conversion in BNS. To determine the molecular mechanisms of fertility conversion at thermosensitive period, we investigated characters of seed setting, anther and pollen grain of fertile and sterile BNS plants and compared young spike proteome patterns at their thermosensitive periods between the two BNS plants. The results showed that sterile plants had lower seed setting rate and pollen number, small pollen grain and lower pollen vitality than fertile plants. Out of protein spots reproducibly detected and analyzed on two dimensional electrophoresis gels, 76 spots showed significant changes in at least one BNS plant and 36 spots were identified by MALDI-TOF MS. The results showed that proteins involved in multiple biochemical pathways were differentially expressed at thermosensitive period between the two plants, including energy metabolism, stress response, signal transduction and regulation, protein process, amino acid and fatty acid metabolism, nucleic acid metabolism etc. Some of these proteins are reported to be involved in the abortion of anther or pollen grains in MS plants, such as energy metabolism and anti oxidative stress, and some were found to be novel proteins involved in the fertility conversion, such as phytohormones regulation. These results indicated that proteins related with anther or pollen development had expressed differently between the two BNS plants before anther development and phytohormones and signal transduction might be involved in the regulation of fertility conversion at thermosensitive period. Our studies have provided new insight to reveal the molecular mechanisms of fertility conversion at thermosensitive period in PTMS wheat.

The application of proteins from less likely studied samples from South East Asia

Protein play multitude of roles in the body of an organism. Enormous effort been done over the years to study proteins either by the tradition al chrumatography techniques of isolating one at a time or by the later developed means of proteomics advances capable to study direc tly on a protein mixture. This paper provides some examples of studies done on either of the mentioned approaches on protein mixtures obtained from samples specifically found predominantly in South East Asia. Expos mes on mapping the two dimensional electrophoresis gel of a number of venom from snakes found commonly in Malaysia and in its neighboring countries will be highlighted. The challenges of mapping protein of abundance, elimination of vertical streaks, lack of protein library, the use of cup loading spiking and 2DE guided purification techniques are some of the important findings. Additionally, the potential development of a protein marker capable to be used to authenticate herbal products incorporated with Tongkat Ali (the notoriously famous aphrodisiac plant) will be also introduced. To conclude the shift of attention from the traditional focus of investigating herbal constituents to that of bioactive protein in natural products rapidly emerging in South East Asia.

Comparison of staining methods for two dimensional electrophoresis gel resolved with <i>Puntius javanicus</i> liver proteome

The aim of this study was to compare the various staining methods based on coomassie briliant blue and silver nitrate stain for the two-dimensional gel electrophoresis resolved with Puntius javanicus liver proteome. The staining methods were selected base on the previous report about their compatibility with the mass spectrometry analysis. Silver staining method is known as the most sensitive method to visualize the maximum number of protein spots resolved in 2D gel but it is less sensitive(incompatible) toward mass spectrometry detection. Results of this study showed that a modified staining method using colloidal coomassie blue G-250 (CCB) is roughly similarly sensitive but lower protein spot detected compared with silver staining (SS) as indicated at the number of 303±26 and 693±14 of protein resolved in both types of stained gels. The conventional methods of staining using coomassie brilliant blue G-250 and R-250 only detected less number of protein spots(128±17and 78±11, respectively) compared to modified CCB staining method. As the coomassie brilliant blue stain is known to be a very sensitive for mass spectrometry detection, the modified method of CCB was selected for further study on Puntius javanicus liver proteome.

A robust protein extraction method for two dimensional electrophoresis of silkworm proteins

Silkworm (Bombyx mori) is a species of agricultural importance, as well as a model organism for Lepidoptera insects. Proteomic method has been widely used in silkworm research, and a robust mass spectrometry-compatible protein extraction method is urgently needed. In this study, we adapted phenol extraction method to extract silkworm midgut protein, and coupled this method with pH 5 - 8 gel strip for two dimensional electrophoresis. The phenol extraction method significantly increased the resolution, as well as greatly reduced the background of two dimensional electrophoresis gels. In addition, this method was well compatible with mass spectrometry analysis. This is the first report that phenol extraction method is used for silkworm midgut protein extraction, and may be applied in other researches.

The role of ezrin-associated protein network in human sperm capacitation.

Membrane modifications in sperm cells represent a key step in sperm capacitation; however, the molecular basis of these modifications is not fully understood. Ezrin is the best-studied member of the ezrin/radixin/merlin family. As a cross-linker between the cortical cytoskeleton and plasma membrane proteins, ezrin contributes to remodeling of the membrane surface structure. Furthermore, activated ezrin and the Rho dissociation inhibitor, RhoGDI, promote the formation of cortical cytoskeleton-polymerized actin through Rho activation. Thus, ezrin, actin, RhoGDI, Rho and plasma membrane proteins form a complicated network in vivo, which contributes to the assembly of the structure of the membrane surface. Previously, we showed that ezrin and RhoGDI1 are expressed in human testes. Thus, we sought to determine whether the ezrin-RhoGDI1-actin-membrane protein network has a role in human sperm capacitation. Our results by Western blot indicate that ezrin is activated by phosphorylation of the threonine567 residue during capacitation. Co-immunoprecipitation studies revealed that, during sperm capacitation, the interaction between ezrin and RhoGDI1 increases, and phosphostaining of two dimensional electrophoresis gels showed that RhoGDI1 is phosphorylated, suggesting that RhoGDI1 dissociates from RhoA and leads to actin polymerization on the sperm head. We speculate that activated ezrin interacts with polymerized actin and the glycosylated membrane protein cd44 after capacitation. Blocking sperm capacitation using ezrin- or actin-specific monoclonal antibodies decreases their acrosome reaction (AR) rate, but has no effect on the AR alone. Taken together, our results show that a network consisting of ezrin, RhoGDI1, RhoA, F-actin and membrane proteins functions to influence the modifications that occur on the membrane of the sperm head during human sperm capacitation.

Automatic lane detection and separation in one dimensional gel images using continuous wavelet transform

This work presents new methods which automatically detect and separate the lanes in one dimensional electrophoresis gel images. The motivation is to present algorithms which can be applied on one dimensional gel images produced from separation of DNA, RNA, (including PCR products), proteins, etc. In addition, a one dimensional intensity profile for each separated lane analogous to capillary electrophoresis (CE) profiles is provided. Experiments are performed on ten multi- and ten single-locus DNA gel images, where the number of lanes, resolution and image quality are highly variable. The Elder & Southern (ES) semi-automatic lane detection method, followed by two new methods: an iterative moving average filter (IMA) and a continuous wavelet transform (CWT) with Morlet wavelet are presented and applied on the test images. The accuracy of the results obtained by applying these methods on a set of 10 multi-locus gel images was 91.7% with the ES, 94.4% with the IMA and 99.5% with the CWT; in the set of 10 single-locus images it was 100% with the ES, 94.8% with the IMA and 97.8% with the CWT; for both sets combined (20 images) it was 96.1% with the ES, 94.7% with the IMA and 98.7% with the CWT. Two lane separation methods are presented; the first is based on a K-neighbouring points algorithm, while a half-width algorithm is applied in the second method. Every separated lane from a gel image can be transferred into a sub-image and further into a one dimensional intensity profile.

Identification and immunolocalization of a 65 kDa drought induced protein in cultivated tomatoLycopersicon esculentum

A 65 kDa protein with a pI value of 5.2 accumulated gradually in tomato leaves during water stress. Protein levels returned to those of the control upon rehydration of the plants. Antiserum raised against the protein, purified from two dimensional electrophoresis gels, was obtained and used as a probe to localize the protein in tomato leaves by immunofluorescence and immunogold labeling. The protein was found to be mainly localized in different areas of nuclei (peripheral chromatin masses, nucleoli and nucleoplasm), chloroplasts, and some leaf cell cytoplasmic regions. Quantification of the gold labeling clearly demonstrated that the amount of the protein increased significantly in nuclei and chloroplasts of cells in drought-stressed plants. Cytological changes occurring in leaf tissues during water stress are also reported.

Age dependent changes in the expression of Drosophila mitochondrial proteins

A subset of 43 [ 35S]methionine labeled mitochondrial proteins, identified on two dimensional electrophoresis gels of whole body extracts of adult Drosophila, were compared between two age groups. These flies, which except for their spermatocytes consist entirely of postmitotic cells, were 6 days old and 38 days old, with the mean life span of this inbred strain being 32.5 days. Qualitative changes in any of these mitochondrial polypeptides were not found, but significant quantitative differences were observed. Quantitation of the 43 mitochondrial proteins was carried out by computer assisted microdensitometry of autoradiograms of the gel patterns. The total amount of isotope incorporated into the mitochondrial proteins of the senescent flies decreased to 71% of that of the young insects. This decrease was heterogeneously distributed among the 43 proteins; however, six remained unchanged and one protein (mol, wt = 75; pl = 7.4) was increased 2.4 times in the senescent flies. These data clearly provide evidence that the age-dependent changes in the expression of mitochondrial proteins are quantitative and not qualitative.