scholarly journals Double-label reductive methylation of tissue proteins for precision two-dimensional polyacrylamide-gel electrophoretic analysis

1981 ◽  
Vol 193 (1) ◽  
pp. 371-374 ◽  
Author(s):  
J M Finger ◽  
K H Choo
Keyword(s):  
High Precision ◽  
Crude Extract ◽  
Gel Electrophoresis ◽  
Electrophoretic Analysis ◽  
Double Labelling ◽  
Two Dimensional ◽  
Reductive Methylation ◽  
Two Dimensional Gel Electrophoresis ◽  
Tissue Samples ◽  
Double Label

Reductive methylation has been used to radioactively label crude-extract proteins with 3H or 14C. The procedure achieved good isotope incorporation and resolution of proteins on two-dimensional polyacrylamide gels. It allows high-precision comparison of tissue samples by double-labelling and should facilitate the study of tissue proteins by two-dimensional gel electrophoresis.

scholarly journals Variations in the Methionine-rich Protein Composition of the Genus Arachis1

1984 ◽  
Vol 11 (1) ◽  
pp. 1-3 ◽  
Author(s):  
Sheikh M. Basha ◽  
Sunil K. Pancholy
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Protein Composition ◽  
Electrophoretic Analysis ◽  
Two Dimensional ◽  
Weight Group ◽  
Two Dimensional Gel Electrophoresis ◽  
One Dimensional ◽  
Molecular Weights ◽  
Isoelectric Points

Abstract Methionine-rich proteins (MRP) from seeds of different species of the Genus Arachis were isolated and analyzed by gel electrophoresis to detect possible compositional differences. One-dimensional gel electrophoretic analysis showed presence of quantitative and qualitative variations among the MRP-fractions. Following two-dimensional gel electrophoresis, the MRP-fractions were found to contain three groups of polypeptides with apparent molecular weights of approximately 21,000; 19,000 and 16,000, and isoelectric points between 5.1 and 5.8. Within each molecular weight group the number of polypeptides varied between 1 and 3.

scholarly journals Two-dimensional gel electrophoretic analysis of the MT3 and DR4 molecules from the different D-typed cells.

1984 ◽  
Vol 160 (3) ◽  
pp. 751-758 ◽  
Author(s):  
M Suzuki ◽  
T Yabe ◽  
M Satake ◽  
T Juji ◽  
H Hamaguchi
Keyword(s):  
Light Chain ◽  
Gel Electrophoresis ◽  
Electrophoretic Analysis ◽  
Structural Heterogeneity ◽  
Class Ii ◽  
Light Chains ◽  
Two Dimensional ◽  
Structural Polymorphism ◽  
Two Dimensional Gel Electrophoresis

This report demonstrates directly, using two-dimensional gel electrophoresis and alloantisera, the following: (a) The DR4 light chains show a structural polymorphism among the Dw4, DKT2, and DYT cells. (b) Most of the class II light chains consist of the DR light chain. (c) The MT3 molecule is distinct from the DR4 molecule in the Dw4, DKT2, and DYT cells. (d) The MT3 molecule does not show any structural heterogeneity among the Dw4, DKT2, and DYT cells. These results suggest that the dissection of the D specificity among Dw4, DKT2, and DYT is mainly caused by the differences of the DR4 molecules.

Effect of Triton X-100 and Dtt Concentrations on Wide Range Two-Dimensional Gel Electrophoresis of Tissue, Cell and Fluid Proteomes

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
Sébastien Charneau ◽  
Gabriel Costa Nunes da Cruz ◽  
Camila Miranda Costa ◽  
Marcelo Valle de Sousa ◽  
Carlos André Ornelas
Keyword(s):  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Water Soluble ◽  
Tissue Cell ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Ph Gradients ◽  
Tissue Samples ◽  
Wide Range ◽  
Triton X

AbstractHigh-resolution separation by two-dimensional gel electrophoresis (2- DE) is still challenging due to the intrinsic behavior of proteins, principally throughout isoelectric focusing separation. It is often observed low resolution of proteins in the alkaline pH region when using wide range pH gradients. Herein, we show the effect of different concentrations of Triton X‑100 and DTT in the sample buffer on wide range pH (3-10) 2-DE profiles of three different biological samples as Trypanosoma cruzi cells, honey bee brain tissue and human saliva fluid. Higher resolution, number and intensity of spots were achieved when 85 mM DTT and 2.5% Triton X‑100 were employed for cell and tissue samples. No improvement was observed for fluid proteins, probably because water-soluble proteins do not require special conditions for extraction and prevention of precipitation during isoelectric focusing.

Muscle protein analysis. III. Analysis of solubilized frozen-tissue sections by two-dimensional electrophoresis.

1981 ◽  
Vol 27 (11) ◽  
pp. 1918-1921 ◽  
Author(s):  
C S Giometti ◽  
N G Anderson
Keyword(s):  
Gel Electrophoresis ◽  
Muscle Protein ◽  
Electrophoretic Analysis ◽  
Human Muscle ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Two Dimensional Electrophoresis ◽  
Tissue Sections ◽  
Frozen Tissue ◽  
Dimensional Electrophoresis

Abstract Proteins from frozen histological sections of human muscle were analyzed by two-dimensional gel electrophoresis. Patterns so obtained were identical to those from whole homogenates of muscle prepared from frozen tissue powders that had much higher protein concentrations. To increase the number of proteins visible on gels of samples low in protein content, the gels were silver stained, or the proteins were labeled with [14C]iodoacetamide before electrophoresis and the gels were fluorographed. The latter method allow use of a single frozen-tissue section for two-dimensional electrophoretic analysis and brings the technique closer to practicable clinical use.

Molecular characterization of an abnormal fibrinogen by two-dimensional electrophoresis.

1984 ◽  
Vol 30 (12) ◽  
pp. 2093-2097 ◽  
Author(s):  
B Polack ◽  
O Valiron ◽  
E Concord ◽  
J M Freyssinet ◽  
G Hudry-Clergeon
Keyword(s):  
Gel Electrophoresis ◽  
Electrophoretic Analysis ◽  
Two Dimensional ◽  
Gamma Chain ◽  
Metrological Analysis ◽  
Two Dimensional Gel Electrophoresis ◽  
Abnormal Rate ◽  
Coomassie Brilliant ◽  
Dimensional Electrophoresis

Abstract We examined normal and abnormal fibrinogen (fibrinogen "Grenoble") by two-dimensional gel electrophoresis to obtain data on possible defects at the molecular level. Fibrinogen Grenoble is characterized by an abnormal rate of fibrin monomer aggregation. The electrophoretic analysis revealed the presence of abnormal gamma chains. Two kinds of gamma chains can be detected in fibrinogen Grenoble: (a) normal gamma chains and (b) gamma chains Grenoble (gamma G) with a greater molecular mass but no modification in isoelectric point. The latter chain can be detected in whole plasma by two-dimensional gel electrophoresis. Metrological analysis was performed in an attempt to quantify observed differences between normal fibrinogen and fibrinogen Grenoble. On use of gels stained either with Coomassie Brilliant Blue or with silver, the partly qualified evaluation gives about 60% normal gamma chain and 40% gamma chain Grenoble.

scholarly journals Mitochondrial ribosome assembly in Neurospora. Two-dimensional gel electrophoretic analysis of mitochondrial ribosomal proteins.

1979 ◽  
Vol 82 (1) ◽  
pp. 17-31 ◽  
Author(s):  
A M Lambowitz ◽  
R J LaPolla ◽  
R A Collins
Keyword(s):  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Protein S ◽  
Electrophoretic Analysis ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Mitochondrial Ribosome

Recent results with Neurospora crassa show that one protein (S-5, mol wt 52,000) associated with the mitochondrial (mit) small ribosomal subunit is translated within the mitochondria (Lambowitz et al. 1976. J. Mol. Biol. 107:223-253). In the present work, Neurospora mit ribosomal proteins were analyzed by two-dimensional gel electrophoresis using a modification of the gel system of Mets and Bogorad. The results show that S-5 is present in near stoichiometric concentrations in high salt (0.5 MKCl)-washed mit small subunits from wild-type strains. S-5 is among the most basic mit ribosomal proteins (pI greater than 10) and has a high affinity for RNA under the conditions of the urea-containing gel buffers. The role of S-5 in mit ribosome assembly was investigated by an indirect method, making use of chloramphenicol to specifically inhibit mit protein synthesis. Chloramphenicol was found to rapidly inhibit the assembly of mit small subunits leading to the formation of CAP-30S particles which sediment slightly behind mature small subunits (LaPolla and Lambowitz. 1977. J. Mol. 116: 189-205). Two-dimensional gel analysis shows that the more slowly sedimentaing CAP-30S particles are deficient in S-5 and in several other proteins, whereas these proteins are present in normal concentrations in mature small subunits from the same cells. Because S-5 is the only mit ribosomal protein whose synthesis is directly inhibited by chloramphenicol, the results tentatively suggest that S-5 plays a role in the assembly of mit small subunits. In addition, the results are consistent with the idea that S-5 stabilizes the binding of several other mit small subunit proteins. Two-dimensional gel electrophoresis was used to examine mit ribosomal proteins from [poky] and six additional extra-nuclear mutants with defects in the assembly of mit small subunits. The electrophoretic mobility of S-5 is not detectably altered in any of the mutants. However, [poky] mit small subunits are deficient in S-5 and also contain several other proteins in abnormally low or high concentrations. These and other results are consistent with a defect in a mit ribosomal constituent in [poky].

Synthesis of an antheridiol-inducible polypeptide during sexual morphogenesis of Achlya ambisexualis E87

1985 ◽  
Vol 63 (5) ◽  
pp. 355-365 ◽  
Author(s):  
J. Stephen Horton ◽  
Paul A. Horgen
Keyword(s):  
Gel Electrophoresis ◽  
Transcriptional Control ◽  
Polyacrylamide Gel Electrophoresis ◽  
Casein Hydrolysate ◽  
Electrophoretic Analysis ◽  
Two Dimensional ◽  
Qualitative Synthesis ◽  
Two Dimensional Gel Electrophoresis ◽  
Achlya Ambisexualis

Changes in in vivo polypeptide synthetic patterns during sexual morphogenesis of Achlya ambisexualis E87 were investigated using polyacrylamide gel electrophoresis. Autoradiography of [35S]methionine-labelled polypeptides revealed the apparent qualitative synthesis of a 64 000 dalton polypeptide, commencing 30–60 min after antheridiol addition and continuing for at least 6 h. Synthesis of this pheromone-inducible polypeptide was detected throughout male sexual morphogenesis in still culture matings of the male strain E87 and the female strain 734. Two other species of Achlya were also found to synthesize the 64 000 dalton polypeptide in response to antheridiol. The inducible polypeptide did not appear to be associated strictly with a branching response, as no detectable synthesis occurred when asexual branching was induced by the exogenous addition of either casein hydrolysate or lactalbumin hydrolysate. Two-dimensional gel electrophoretic analysis revealed quantitative changes in the synthesis of many other polypeptides as a result of antheridiol action. In addition, both equilibrium and nonequilibrium two-dimensional gel electrophoresis showed the inducible polypeptide to be basic, demonstrating an isoelectric point of more than 8. Studies using the RNA synthetic inhibitor actinomycin D suggested a transcriptional control of the inducible polypeptide.

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