Analysis of the cytosolic proteins of chick embryo gonads by two-dimensional gel electrophoresis

Development ◽  
1986 ◽  
Vol 94 (1) ◽  
pp. 221-230
Author(s):  
J. Samsel ◽  
B. Lorber ◽  
A. Petit ◽  
J. -P. Weniger
Keyword(s):  
Gel Electrophoresis ◽  
Developmental Stages ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Protein ◽  
Chick Embryos ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Specific Proteins ◽  
Different Tissues

Gonads and mesonephros dissected from normal chick embryos, as well as a blood sample, were labelled in vitro with [35S]methionine and [14C]leucine. The patterns of cytosolic protein synthesis of the different tissues were analysed using two-dimensional (2-D) polyacrylamide gel electrophoresis. Three developmental stages, i.e. 8, 12 and 17 days of incubation, were investigated. Five sex-specific proteins were detected in the male and two in the female. In the testis, only one protein is already present at the 8-day stage. In the ovary, one protein exists since the 8-day stage, but it is also synthesized in the mesonephros. The second ovary-specific protein appeared only at the 12-day stage.

scholarly journals Clustering of Clinical Strains ofHelicobacter pylori Analyzed by Two-Dimensional Gel Electrophoresis

2000 ◽  
Vol 7 (2) ◽  
pp. 301-306 ◽  
Author(s):  
Helena Enroth ◽  
Thomas Åkerlund ◽  
Anna Sillén ◽  
Lars Engstrand
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Two Dimensional ◽  
Strain Variation ◽  
Disease Group ◽  
Two Dimensional Gel Electrophoresis ◽  
Specific Proteins ◽  
Patient Groups ◽  
H Pylori ◽  
Disease Specific

ABSTRACT Strain variations of Helicobacter pylori have been tested by numerous methods and compared among different patient groups. The aim of this study was to investigate whether H. pyloriexpresses disease-specific proteins that can be detected by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). H. pylori strains isolated from duodenal ulcer, gastric cancer, and gastritis patients were analyzed. Extensive variation in spot patterns was observed between the strains, but a dendrogram analysis revealed that some strains within each disease group clustered together. Eight proteins were sequenced and found in the H. pylori genome sequence. 2-D PAGE is a useful method for studies of protein expression and for highlighting the extensive strain variation that H. pylori exhibits.

scholarly journals Dog pancreatic microsomal-membrane polypeptides analysed by two-dimensional gel electrophoresis

1984 ◽  
Vol 217 (1) ◽  
pp. 145-157 ◽  
Author(s):  
M A Kaderbhai ◽  
B M Austen
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Microsomal Membrane ◽  
Translation System ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Microsomal Preparation ◽  
Microsomal Membranes

The two-dimensional polyacrylamide-gel electrophoresis technique of O'Farrell [(1975) J. Biol. Chem 250, 4007-4021] was applied to resolve and analyse the polypeptide composition of dog pancreatic rough microsomal membranes, which were shown to be active in co-translational processing of preprolactin synthesized from pituitary mRNA in a translation system in vitro. About 100 polypeptides are resolved. Treatment of rough microsomal membranes with EDTA and high KCl concentration yielded membranes stripped of their ribosomes with retention of activity for translocation and processing. Stripped microsomal membranes showed a selective concentration of approximately 25 polypeptides in the membranes when analysed by two-dimensional polyacrylamide-gel electrophoresis. The two-dimensional electrophoretic profile was catalogued into polypeptides that are glycoproteins, those that contain free thiol groups disposed at the cytosolic surface of microsomal vesicles and those that are of secretory origin but have been entrapped in the microsomal preparation. Several secretory components, including amylase, procarboxypeptidases, lipase and anionic trypsinogen, were tentatively identified among the microsomal polypeptides. The rough and stripped microsomal membranes from dog pancreas show a characteristic set of seven major acidic polypeptides, which are also identifiable in microsomal-membrane preparations isolated from dog liver and rat liver. One of these polypeptides was identified as protein disulphide-isomerase (EC 5.3.4.1).

scholarly journals The SPS4 gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA.

1986 ◽  
Vol 6 (12) ◽  
pp. 4478-4485 ◽  
Author(s):  
A T Garber ◽  
J Segall
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Protein ◽  
Polyacrylamide Gel ◽  
In Vitro Translation ◽  
Specific Gene ◽  
Alpha Cells ◽  
Two Dimensional ◽  
Isoelectric Ph

The SPS4 gene of Saccharomyces cerevisiae, a sporulation-specific gene identified previously in a differential hybridization screen of a genomic yeast DNA library, has been characterized further. The protein encoded by this gene was inferred from its nucleotide sequence to be 38,600 daltons with an isoelectric pH of 8.2. Consistent with this, two-dimensional polyacrylamide gel electrophoresis of the in vitro translation products of RNA purified by hybridization with the cloned SPS4 DNA indicated that the SPS4 gene product is a 39-kilodalton, basic protein. This protein was found to be identical in size and charge to a major, sporulation-specific protein identified in a two-dimensional polyacrylamide gel electrophoretic comparison of the in vitro translation products of total RNA from sporulating MATa/MAT alpha cells and asporogenous MAT alpha/MAT alpha cells. A MATa/MAT alpha strain homozygous for a partial deletion of the SPS4 gene appeared, however, to be unaffected in its ability to form viable ascospores.

scholarly journals Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

1981 ◽  
Vol 91 (2) ◽  
pp. 352-360 ◽  
Author(s):  
TW McKeithan ◽  
JL Rosenbaum
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Cold Temperature ◽  
Two Dimensional ◽  
Multiple Forms ◽  
Cytoplasmic Fraction ◽  
Two Dimensional Gel Electrophoresis ◽  
Brain Tubulin ◽  
Β Tubulin

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.

scholarly journals Isolation from human chronic-lymphocytic-leukaemia cells of membrane glycoproteins associated with Fc-receptor functions. Physical parameters and production of polyclonal antibodies

1987 ◽  
Vol 245 (1) ◽  
pp. 75-83 ◽  
Author(s):  
G Gorini ◽  
G A Medgyesi ◽  
M Garavini ◽  
K J Dorrington ◽  
J Down
Keyword(s):  
Chronic Lymphocytic Leukaemia ◽  
Gel Electrophoresis ◽  
Lymphocytic Leukaemia ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fc Receptor ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Membrane Glycoproteins

Two membrane glycoproteins that bound immune complexes and inhibited Fc-receptor- (FcR-)mediated functions in vitro were purified from human FcR+ chronic-lymphocytic-leukaemia cells. A multi-step purification was developed, consisting essentially in: (i) Tween 40 extraction of crude cell membranes; (ii) solubilization of membrane fragments by Renex-30; (iii) isolation of glycoproteins by affinity chromatography on Lens culinaris haemagglutinin-Sepharose; (iv) papain treatment of the eluted glycoproteins followed by gel-filtration chromatography; (v) purification by polyacrylamide-gel electrophoresis of two molecular species from the protein-size fraction enriched for immune-complex-binding activity. The two electrophoretically isolated components displayed apparent molecular masses of 70 and 45 kDa by SDS/polyacrylamide-gel electrophoresis and restricted charge heterogeneity by two-dimensional analysis. Two-dimensional peptide mapping revealed the presence of many peptides in common between the two proteins and the absence of a number of peptides in the 45 kDa component. These two polypeptides were used as immunogens to produce polyclonal antibodies that cross-reacted with both proteins and specifically inhibited FcR-mediated reactions in vitro. Furthermore, FcR-related components from detergent-extracted lysates of the human K562 and U937 cell lines or human placental membranes were revealed by the putative anti-FcR antibodies adsorbed on Protein A-Sepharose.

A New Method for Proteome Screening with Two-Dimensional Urea-Sds Polyacrylamide Gel Electrophoresis

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
Areeba Ahmad ◽  
Riaz Ahmad
Keyword(s):  
Gel Electrophoresis ◽  
Protein Separation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cost Effective ◽  
Polyacrylamide Gel ◽  
Present System ◽  
Liver Protein ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis

AbstractTwo-dimensional gel electrophoresis (2DE) separating proteins on the basis of their pI and molecular mass remain the best available technique for protein separation and characterization to date. But due to several limitations, including streak formation in IEF gels, partial solubility of proteins, expensive running conditions and relatively longer time taken, a simple urea-SDS-2D polyacrylamide gel electrophoresis (US2DE) is described here. The system is reasonably sensitive, cost effective with good reproducibility. The method described in this paper employs a chaotropic agent, urea, in the first dimension and sodium dodecyl sulphate (SDS), like conventional system, in the second dimension with an addition of polyacrylamide to screen the liver proteome of healthy and chemically induced fibrotic rats. The system separates the protein on the basis of chargeto- mass ratio and clearly demonstrates differential expression in the liver protein repertoire of healthy and fibrotic rats. Moreover, the present system, like other 2D electrophoretic procedures revealed at least 22 novel spots in the investigated tissues. The technique may be utilized for comprehensive proteome screening of any biological sample and would provide an overview to narrow down the candidate proteins or biomarkers.

Fibrin-I Degradation Products with a Molecular Weight Higher than That of Fibrinogen

1973 ◽  
Vol 29 (01) ◽  
pp. 122-129 ◽  
Author(s):  
René von Hugo ◽  
Henner Graeff
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Degradation Products ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Two Dimensional ◽  
Plasma Clot ◽  
Two Dimensional Gel Electrophoresis ◽  
Parent Molecule

SummaryThe observation of intravascular lysis of fibrin deposits and of fibrinogen derivatives with a molecular weight higher than the parent molecule in human cases of disseminated intravascular coagulation (DIC) initiated the following in vitro study. Following streptokinase induced plasma clot solubilization fibrinogen derivatives were investigated after ß-alanine precipitation of the plasma samples by polyacrylamide (PAA) gel electrophoresis, intra gel immunoprecipitation, two dimensional gel electrophoresis and by agarose gel filtration. Three fibrin-i degradation products were observed and characterized according to their relative electrophoretic mobility in 5% PAA gel: 0.23, 0.35, 0.46 (fibrinogen: 0.43) x 10-5 cm2/V x sec. They could also be demonstrated after electrophoresis in the presence of 5 M urea. Agarose gel filtration yielded one peak at 180 ml of effluent volume. The 0.23 derivative was eluted in the peak fractions, whilst the 0.35 and 0.46 derivatives were eluted together at approximately 201 ml of the effluent volume (fibrinogen: 225 ml). This indicates, that the three fibrin-i degradation products described are molecular entities with molecular weights higher than fibrinogen and, that the 0.46 derivative has an increased charge/molecular size ratio in comparison with fibrinogen. Corresponding data were obtained by two dimensional gel electrophoresis in gels of different pore size.

scholarly journals Proteomic Characterisation of Lupin (Lupinus angustifolius) Milk as Influenced by Extraction Techniques, Seed Coat and Cultivars

Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1782
Author(s):  
Nadia Al-Saedi ◽  
Manjree Agarwal ◽  
Wujun Ma ◽  
Shahidul Islam ◽  
Yonglin Ren
Keyword(s):  
Gel Electrophoresis ◽  
Milk Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Two Dimensional ◽  
Extraction Techniques ◽  
Centrifuge Method ◽  
Lupin Seeds ◽  
Specific Proteins ◽  
Protein Extractability ◽  
Better Than

Lupin seeds are rich in proteins and other essential ingredients that can help to improve human health. The protein contents in both whole and split seeds of two lupin cultivars (Mandleup and PBA Jurien) were used to produce the lupin milk using the cheesecloth and centrifuge method. Proteins were extracted from the lupin milk using thiourea/urea solubilization. The proteins were separated by a two-dimensional polyacrylamide gel electrophoresis and then identified with mass spectrometry. A total of 230 protein spots were identified, 60 of which showed differential abundances. The cheesecloth separation showed protein extractability much better than that of the centrifuge method for both the cultivars. The results from this study could offer guidance for future comparative analysis and identification of lupin milk protein and provide effective separation technique to determine specific proteins in the cheese-making process.

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