Two-dimensional gel electrophoresis of serum specimens from a normal population.

1982 ◽  
Vol 28 (4) ◽  
pp. 890-899 ◽  
Author(s):  
R P Tracy ◽  
R M Currie ◽  
D S Young
Keyword(s):  
Gel Electrophoresis ◽  
Serum Proteins ◽  
Protein Pattern ◽  
Normal Population ◽  
Small Samples ◽  
Two Dimensional ◽  
Silver Stain ◽  
Two Dimensional Gel Electrophoresis ◽  
Finger Stick ◽  
Effects Of Aging

Abstract We examined sera from a normal population by two-dimensional gel electrophoresis, to establish the normal pattern of serum proteins and to investigate genetic polymorphisms. With such information in hand, specimens from patients with certain diseases may be readily evaluated. Towards this goal, we optimized the ISO-DALT system (Proc. Natl, Acad, Sci, USA 74: 5421--5425, 1977) for routine phenotyping of alpha 1-antitrypsin, haptoglobin, GC-globulin, alpha 2-HS-glycoprotein, and transferrin, as well as a previously unknown polymorphic protein. We examined the effects of aging the specimens for 2 h at room temperature (no changes) or at -20 degrees C for several months (small changes), as well as serum/plasma differences and the effect of protease inhibitors. Silver-stain methods were modified to allow simultaneous staining of 10 gels, with reasonably good reproducibility of stain intensity. We quantitated silver-stained gels by densitometry of photographic transparencies. Very small samples suffice with this stain (0.5 microL of serum or plasma), allowing the use of "finger-stick" methods instead of venipuncture, yet the patterns are better resolved and easier to read than those for 10-microL specimens processed on gels stained with Coomassie Blue. Our techniques for rapidly removing albumin and IgG allow the investigator to examine areas on the gel that ordinarily are obscured. The region of haptoglobin has been examined by using serum from an ahaptoglobinemic donor. Finally, we present an expanded "normal" map illustrating the composite protein pattern.

Reproducibility and quality assurance of two-dimensional gel electrophoresis of serum specimens.

1982 ◽  
Vol 28 (4) ◽  
pp. 908-914 ◽  
Author(s):  
R P Tracy ◽  
R M Currie ◽  
D S Young
Keyword(s):  
Quality Assurance ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Two Dimensional ◽  
Protein Loss ◽  
Serum Samples ◽  
Silver Stain ◽  
Two Dimensional Gel Electrophoresis ◽  
Report Quality ◽  
Wide Range

Abstract Currently we are using two different ISO-DALT two-dimensional gel electrophoresis systems, designated MC-Iso 1 and MC-Iso 2, for the analysis of serum and plasma samples. Here we report quality-assurance data for both of these systems. CV values for the slopes of the pH gradient (ISO dimension) are 5.6% of less; CV values for the slopes of the molecular-mass curves (log Mr vs relative mobility in the DALT dimension) are 3.4% or less. We examined the various steps of the analysis in detail for reproducibility and protein loss, using radiolabeled albumin, alpha 2-macroglobulin, and beta 2-microglobulin. Generally, in the first dimension, less protein enters the MC-Iso 2 gels (our routine system in which silver stain is used) than enters the MC-Iso 1 gels (our wide-range system for myeloma serum samples, in which the gel is stained with Coomassie Blue), on the average, 87% as much. The CV at this stage for both systems is 5--8%. During equilibration, considerable amounts of protein are lost (approximately 30% in 10 min) from the ISO gel, and the reproducibility is also decreased. Resolution in the DALT dimension has, in most cases, little or no effect on either recovery or reproducibility. Overall, for most proteins expected to appear in an ISO gel of a given pH range, approximately 50--60% of the starting material may be expected to reside in the sodium dodecyl sulfate slab gel, under our conditions. The two most important variables affecting recovery are the concentration of the NaOH (used as catholyte) and the pH of the starting sample. The overall CV for the process is between 8 and 12%.

Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis.

1982 ◽  
Vol 28 (4) ◽  
pp. 955-961 ◽  
Author(s):  
C S Giometti ◽  
K E Willard ◽  
N L Anderson
Keyword(s):  
Gel Electrophoresis ◽  
Lymphoblastoid Cell Line ◽  
Protein Pattern ◽  
Cytoskeletal Proteins ◽  
Cytoskeletal Protein ◽  
Two Dimensional ◽  
Human Skin Fibroblasts ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Two Dimensional Gel Electrophoresis

Abstract Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. Three groups (Cytoskf:8--10, :14--16, and :17--18) showed qualitative differences, and the fourth group (Cytoskf:11 and :13) showed quantitative differences. In addition, surface labeling of GM607 and human fibroblasts with 125I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. Cytoskf:11 and :13 in fibroblast preparations were also labeled with the 125I. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture.

Effects of selenium deficiency on the rat myocardial protein pattern - investigation by two-dimensional gel electrophoresis

2000 ◽  
Vol 95 (3) ◽  
pp. 199-207 ◽  
Author(s):  
Vera Regitz-Zagrosek ◽  
Antonius Kyriakopoulos ◽  
Peter Jungblut ◽  
Dietrich Behne ◽  
Klaus-Peter Plei�ner ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Protein Pattern ◽  
Selenium Deficiency ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis

Changes in patterns of protein synthesis in axolotl oocytes during progesterone-induced maturation

Development ◽  
1986 ◽  
Vol 92 (1) ◽  
pp. 103-113
Author(s):  
Jean Gautier ◽  
Renée Tencer
Keyword(s):  
Protein Synthesis ◽  
Protein Phosphorylation ◽  
Cholera Toxin ◽  
Oocyte Maturation ◽  
Gel Electrophoresis ◽  
De Novo ◽  
Protein Pattern ◽  
Ambystoma Mexicanum ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis

Patterns of protein phosphorylation and synthesis during axolotl (Ambystoma mexicanum) oocyte maturation were studied by incorporation of [32P]orthophosphate and [35S]methionine into polypeptides, followed by two-dimensional gel electrophoresis. Various alterations were observed after progesterone treatment: de novo appearance of [35S]methionine-labelled polypeptides, a quantitative increase in previously synthesized proteins and a quantitative decrease in or disappearance of other previously synthesized proteins. Changes in 32P- and 35S-labelling were observed very early during maturation. Neither prior oocyte enucleation nor α-amanitin treatment had a significant effect on these changes. Stimulation with MPF provided the same final protein pattern as PG treatment. However, cholera toxin inhibited all the changes seen during maturation. Comparisons between the patterns of [35S]methionine- and [32P]phosphatelabelling provide further information on the biochemical events that take place during oocyte maturation.

Improved two-dimensional gel electrophoresis representation of serum proteins by using ProtoClear™

2007 ◽  
pp. 274-279
Author(s):  
Bridget A. Lollo ◽  
Sheryl Harvey ◽  
Jane Liao ◽  
Anthony C. Stevens ◽  
Raymond Wagenknecht ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Serum Proteins ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis of cerebrospinal fluid proteins.

1980 ◽  
Vol 26 (9) ◽  
pp. 1317-1322 ◽  
Author(s):  
D Goldman ◽  
C R Merril ◽  
M H Ebert
Keyword(s):  
Cerebrospinal Fluid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Spinal Fluid ◽  
Two Dimensional ◽  
Silver Stain ◽  
Two Dimensional Gel Electrophoresis ◽  
Cerebrospinal Fluid Proteins ◽  
High Resolution Analysis

Abstract Two-dimensional electrophoresis, with isoelectric focusing in the first dimension and sodium dodecyl sulfate/polyacrylamide gel electrophoresis in the second, has been adapted for the high-resolution analysis of cerebrospinal fluid proteins. Proteins were detected with a new, highly sensitive silver stain that made visible more than 300 polypeptides from 60 microL of spinal fluid, in highly reproducible patterns. We have mapped these patterns, noting difference between the proteins observed in spinal fluid and plasma, and have prepared a partial map of cerebrospinal fluid proteins.

Proteins in normal, irradiated, and postmortem human brain quantitatively compared by using two-dimensional gel electrophoresis.

1984 ◽  
Vol 30 (12) ◽  
pp. 1989-1995 ◽  
Author(s):  
R K Narayan ◽  
W E Heydorn ◽  
G J Creed ◽  
P L Kornblith ◽  
D M Jacobowitz
Keyword(s):  
Cerebral Cortex ◽  
Gel Electrophoresis ◽  
Protein Pattern ◽  
Relative Molecular Mass ◽  
Two Dimensional ◽  
Protein Patterns ◽  
Human Cerebral Cortex ◽  
Postmortem Human Brain ◽  
Two Dimensional Gel Electrophoresis ◽  
Fresh Frozen

Abstract Using a combination of two-dimensional gel electrophoresis (2DE), silver staining, and computerized densitometry, we studied protein patterns in human cerebral cortex: normal fresh-frozen, fresh-frozen but previously irradiated, and post-mortem. The relative molecular mass of the resolved proteins ranged from 14 400 to 100 000, the isoelectric points from 4.75 to 7.0. The pattern of proteins (six of them identified) was essentially the same for all three groups. However, computerized densitometry demonstrated significant alterations in the density of several spots in the irradiated and postmortem groups as compared with the normal controls. Irradiated cortex showed statistically significant changes in only six spots (three increased and three decreased in density); postmortem material showed 20 altered spots (16 diminished and four increased). Evidently normal human cerebral cortex has a consistent protein pattern on 2DE, which is quantitatively (but not qualitatively) altered in irradiated and postmortem material. These findings provide a point of reference against which proteins from abnormal brain material can be compared, both qualitatively and quantitatively.

scholarly journals Analysis of proteins associated with storage root formation in cassava using two-dimensional gel electrophoresis

2001 ◽  
Vol 13 (1) ◽  
pp. 41-48 ◽  
Author(s):  
GLAUCIA B. CABRAL ◽  
LUIZ J.C.B. CARVALHO
Keyword(s):  
Gel Electrophoresis ◽  
Adventitious Root ◽  
Protein Pattern ◽  
Protein Profile ◽  
Secondary Growth ◽  
Starch Accumulation ◽  
Computer Assisted ◽  
Two Dimensional ◽  
Storage Root ◽  
Two Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis analysis was performed on adventitious and storage root in cassava (Manihot esculenta Crantz). Adventitious root lacking swelling formation and swelled storage root were obtained from the accession WU104 grown in the green house of the Department of Biology in Washington University in St. Louis (MO-USA). Saline buffer-soluble proteins were extracted, separated in a high-resolution 2-D electrophoresis system, visualized with silver staining gel procedure, and digital image generate for further analysis. Quantitative and qualitative protein spots analysis was performed with a computer assisted image software system. Results revealed large variation in the complexity of the gel protein profile between the two root systems. About 90% of the protein spots appeared in the pI range value of 4.0 to 6.5 and between 14 to 80 Kda of molecular mass. Detailed computer assisted analysis of this gel allowed us to establish 5 distinct classes of protein based on spot quantification that could be associated with swelling and non-swelling roots. Variation in the complexity of protein pattern was related with different type of root. Whereas the adventitious root showed a more simple profile related to primary growth, the storage root showed to be a more complex profile related to secondary growth and starch accumulation.

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