Detection of spatially- and stage-specific proteins in extracts from single embryos of the domesticated carrot

Development ◽  
1988 ◽  
Vol 103 (4) ◽  
pp. 665-674
Author(s):  
R.H. Racusen ◽  
F.M. Schiavone
Keyword(s):  
Developmental Stages ◽  
Surgical Techniques ◽  
Protein Separations ◽  
Two Dimensional ◽  
Protein Distribution ◽  
Two Dimensional Electrophoresis ◽  
Sds Page ◽  
Spatial Differences ◽  
Specific Proteins ◽  
Dimensional Electrophoresis

Single embryos, representing each of four distinct morphological stages, were selected from cultures of the domesticated carrot for analysis of total [35S]methionine-labelled proteins. Following exposure to radiolabel for 12 to 18h, embryos were individually disrupted in a 3mm diameter, precisely-matched, plastic mortar and pestle. Radiolabelled proteins extracted by this procedure were separated by two-dimensional electrophoresis procedures, consisting of isoelectric focusing in 1mm tubes, followed by SDS-PAGE in a small slab gel. Comparisons of autoradiographs of these gels revealed that the levels of a number of proteins were modulated during the conversion of disordered callus cells into maturing embryos. In addition, miniature surgical techniques were used to separate the apex (cotyledon end) from the base (root end) of late-stage embryos, for extraction of proteins and analysis of spatial differences in protein distribution. About five proteins in extracts from each section were observed to be synthesized at different rates in the two halves, indicating that there are molecular correlates for early polarized growth. About half of the proteins, whose appearances were unique to apical and basal sections of embryos, were also observed to fluctuate in comparisons of autoradiographs of two-dimensional protein separations from embryos at different developmental stages.

Comparison of protein composition between schistosomula of Schistosoma japonicum and S. mansoni

Parasitology ◽  
1984 ◽  
Vol 89 (3) ◽  
pp. 453-459 ◽  
Author(s):  
S. Maeda ◽  
Y. Irie ◽  
K. Yasuraoka
Keyword(s):  
Schistosoma Japonicum ◽  
Protein Composition ◽  
Two Dimensional ◽  
Japanese Strain ◽  
Two Dimensional Electrophoresis ◽  
Sds Page ◽  
Specific Proteins ◽  
Protein Components ◽  
Dimensional Electrophoresis

SUMMARYSDS-PAGE analysis revealed that the protein composition of mechanically transformed schistosomula (ms) of a Japanese strain of Schistosoma japonicum was essentially similar to that of a Philippine strain of S. japonicum. However, the protein components of S. mansoni ms were remarkably different from those of S. japonicum. Specific proteins of Mr 60–65.and 30 kDa were found in ms of S. japonicum and S. mansoni, respectively. In skin-penetrated schistosomula (ss) of the two species, a common protein of 67 kDa was detected. Using two-dimensional electrophoresis, strain-specific polypeptides (9 in a Japanese and 11 in a Philippine strain) were identified in ms of S. japonicum. Mechanically transformed and skin-penetrated schistosomula of S. japonicum (Japanese) had, respectively, 7 and 10 specific proteins. Four and 1 specific polypeptides were also detected in two-dimensional profiles of ms and ss of S. mansoni, respectively.

scholarly journals Platelet Membrane Actin and Myosin

1977 ◽  
Author(s):  
H. Horák ◽  
P.G. Barton ◽  
C.M. Gibbs
Keyword(s):  
Cell Membrane ◽  
Membrane Vesicles ◽  
Two Dimensional ◽  
Con A ◽  
Whole Cells ◽  
Two Dimensional Electrophoresis ◽  
Membrane Surfaces ◽  
Sds Page ◽  
Inside Out ◽  
Dimensional Electrophoresis

Horse platelet membranes isolated by the glycerol lysis technique and subjected to SDS-PAGE showed large amounts of actin and variable amounts of myosin relative to other membrane proteins and glycoproteins. [14C]-2-dinitrothioadenosine diphosphate, when briefly incubated with whole cells, rapidly labeled the membrane actin component. Retention of myosin by the membranes during their isolation was optimized by lysing the cells and resuspending the membranes in Tris-HC1, pH 7.35, with 0.13 M KCl, 0.01 M NaCl, 2 mM MgCl2 and 0.01 mM CaCl2. Subsequently, significant amounts of actin and myosin could be eluted from the membranes with 10-3 M ADP but not with CDP, GDP or UDP. Actin was also eluted effectively from membranes prepared in Tris-NaCl, pH 7.35, by washing with 0.1 mM EDTA (in presence or absence of ADP). Despite repeated washings with either elution system, more than 50% of the actin remained associated with the membranes. When membrane vesicles with right side out (RO) and inside out (IO) orientation, separated by chromatography on Con A-Sepharose, were similarly washed identical results were obtained. Two dimensional electrophoresis of the membrane protein of IO vesicles separated two major actin components, one of which was differentially removed by prior treatment of the vesicles with 10-3 M ADP. It is concluded that (1) platelet myosin and two forms of platelet actin are associated with the cell membrane and that myosin and one form of actin can be displaced by ADP or EDTA while the second form of actin is more firmly attached, and (2) some actin is present on both membrane surfaces.

scholarly journals Physical evaluation, morphological and identification of seminal proteins in Santa Ines sheep

2017 ◽  
Vol 18 (1) ◽  
pp. 211-220
Author(s):  
Marilza Assunção de OLIVEIRA ◽  
Roseane Pinto Martins de OLIVEIRA ◽  
Ana Rita de LIMA ◽  
Edmar Vaz de ANDRADE ◽  
Jan Sidarta Lima de ABREU ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Markers ◽  
Seminal Plasma ◽  
Two Dimensional ◽  
Two Dimensional Electrophoresis ◽  
Physiological Processes ◽  
Zeta Chain ◽  
Sds Page ◽  
Santa Inês ◽  
Dimensional Electrophoresis

SUMMARY This study aimed to identify proteins in the seminal plasma associated with fertility in sheep of Santa Inês in Manaus, AM, using twodimensional electrophoresis techniques associated with mass spectrometry. Semen samples from eight adult sheep were collected by removing an aliquot for the physical and morphological assessments of semen and seminal plasma was subjected to SDS-PAGE profile and two-dimensional electrophoresis. Gels were stained with colloidal Coomassie, scanned and analyzed using ImageMaster 2D Platinum software, version 6.0. The selected individual spots were cut from the master gel, digested with trypsin and subjected to identification by mass spectrometry (MALDITof / Tof). Of the 108 spots detected in the gel, it selected 10 differential spots (based on the distribution thereof in the bidimensional gel and pre-analysis of the 2D ImageMaster Platinum Software) identifying 03 proteins: clusterin, a protein 14-3-3 zeta chain and Ram Seminal versicles 22kDa Protein. The identity of these proteins implies that the components of seminal plasma participate in physiological processes involved in sperm protection, motility and sperm capacitation, all associated with fertility. These proteins need to be better studied to see whether the same could be used as molecular markers of fertility as they were also found in other studies conducted with sheep Santa Ines.

Microscale multisample two-dimensional electrophoresis of proteins in human serum, cerebrospinal fluid, and urine.

1982 ◽  
Vol 28 (4) ◽  
pp. 824-827 ◽  
Author(s):  
T Manabe ◽  
E Hayama ◽  
T Okuyama
Keyword(s):  
Cerebrospinal Fluid ◽  
Human Serum ◽  
Distribution Patterns ◽  
Blue Staining ◽  
Two Dimensional ◽  
Protein Distribution ◽  
Two Dimensional Electrophoresis ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Dimensional Electrophoresis

Abstract In this technique, in which no denaturing agent is used, proteins in human serum, cerebrospinal fluid, and urine are separated by isoelectric focusing in cylindrical 40 g/L polyacrylamide gels of capillary size (1.3 x 35 mm) for 40 min, followed by electrophoresis in 40--170 g/L polyacrylamide linear gradient gel, with use of 38 x 35 x 1 mm slab gel, for 1 h. Only 2 microL of untreated human serum is required to obtain clear protein-distribution patterns, made visible by Coomassie Blue staining. By use of silver staining, proteins in unconcentrated cerebrospinal fluid can be made visible. An apparatus we devised for microscale two-dimensional electrophoresis enables us to analyze eight protein samples simultaneously.

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