scholarly journals Detection in milk of a 60,000 Mr mouse and a 68,000 Mr human phosphorylated glycoprotein by histochemical analysis on polyacrylamide gels.

1983 ◽  
Vol 31 (6) ◽  
pp. 709-716 ◽  
Author(s):  
M R Green
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Blue Staining ◽  
Polyacrylamide Gels ◽  
Periodic Acid Schiff ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Proteins in colostrum and skimmed milk from humans and mice were separated by electrophoresis on polyacrylamide gels and stained with Coomassie blue (CB), Ethyl-Stains-all (ESA), and periodic acid-Schiff (PAS) to investigate changes that may occur in milks throughout lactation. In mouse colostrum but not in mature mouse milk, a PAS-positive protein of apparent molecular weight of 60,000 stained prominently blue with ESA. A protein in human milk with a molecular weight of 68,000 stained similarly but was present throughout lactation. The intensity of blue staining of these minor proteins in milk approached that obtained with casein phosphoproteins. The metachromatic dye ESA stains phosphoproteins and sialic acid-rich glycoproteins blue to blue-green. Removal of phosphorus from the former and sialic acid from the latter results in those proteins staining red with ESA. The intensity of blue staining of the 60,000 and 68,000 Mr proteins was diminished but not lost following treatment with phosphatase. It was eliminated following neuraminidase digestion of the mouse protein and mild acid hydrolysis of the human protein. Coomassie blue staining of the proteins was not affected by these procedures. Following electrophoresis of milk and milk fractions in a non-sodium dodecyl sulfate-containing system, the proteins were identified by their characteristic staining properties with ESA and isolated.

Oviductal fluid protein patterns in the rhesus monkey (Macaca mulatta) during the menstrual cycle

1992 ◽  
Vol 4 (2) ◽  
pp. 249 ◽  
Author(s):  
A Paliwal ◽  
B Malaviya ◽  
VP Kamboj
Keyword(s):  
Menstrual Cycle ◽  
Protein Profile ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Blue Staining ◽  
Protein Patterns ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Oviductal Fluid

Oviducts were obtained from monkeys on Days 8, 14, 19 and 25 of the menstrual cycle and changes in the pattern of luminal fluid proteins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis after periodic acid Schiff's reagent (PAS) and coomassie blue staining of the gels revealed 85 and 95 kDa proteins only up to Day 14 whereas a 130 kDa glycoprotein persisted up to Day 19 and reached a nadir at mid-menstrual cycle (Day 14). The absence of the 130 kDa glycoprotein in the serum and its presence in cytosolic preparations up to Day 19 suggest that it is of oviductal origin. The 130 kDa glycoprotein is of particular interest since it was present in the oviductal fluid during mid cycle, a period when the oviduct participates in gamete transport, fertilization and embryo development. The conclusion drawn from this study is that the protein profile of monkey oviductal fluid changes during the menstrual cycle.

scholarly journals Characterization of a high-molecular-weight glycoprotein isolated from the pulmonary secretions of patients with alveolar proteinosis

1979 ◽  
Vol 177 (1) ◽  
pp. 153-158 ◽  
Author(s):  
Saura Sahu ◽  
William S. Lynn
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Periodic Acid Schiff ◽  
Major Band ◽  
Coomassie Blue ◽  
Neutral Sugars ◽  
Alveolar Proteinosis

A high-molecular-weight glycoprotein was isolated, purified and partially characterized from the insoluble pulmonary secretions accumulating in lungs of patients suffering from pulmonary alveolar proteinosis. On electrophoresis in 5% polyacrylamide gel in the presence of sodium dodecyl sulphate and 2-mercaptoethanol, the purified protein gave one major band as detected by Coomassie Blue as well as with periodic acid/Schiff staining. An apparent mol.wt. of 250000 was estimated for this glycoprotein. Amino acid analysis showed that it contains hydroxyproline, and relatively high amounts of glycine, glutamic acid, aspartic acid and leucine. It contains approx. 6% hexose, 3% sialic acid and 2% glucosamine. The neutral sugars are galactose, mannose and fucose. An antiserum prepared in rabbits against this high-molecular-weight glycoprotein cross-reacted with two smaller glycoproteins (mol.wts. 62000 and 36000) isolated from the same pulmonary secretions of these patients. A complementary observation was also made when this large alveolar glycoprotein cross-reacted with an antiserum prepared in rabbits against the smaller glycoprotein (mol.wt. 36000). It appears that this high-molecular-weight glycoprotein may be the precursor of the two smaller glycoproteins present in the same diseased pulmonary secretions.

scholarly journals Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain

1976 ◽  
Vol 156 (1) ◽  
pp. 143-150 ◽  
Author(s):  
R H Quarles
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Weight Class ◽  
Developmental Difference ◽  
Adult Rats ◽  
Neuraminidase Treatment

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.

scholarly journals Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1212-1219 ◽  
Author(s):  
N Kieffer ◽  
B Boizard ◽  
D Didry ◽  
JL Wautier ◽  
AT Nurden
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Blue Staining ◽  
Igg Antibody ◽  
Immune Disorder ◽  
Immunochemical Characterization ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Abstract We report the immunochemical characterization of a new platelet- specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.

scholarly journals Purification, characterization and radioimmunoassay of adenosine deaminase from human leukaemic granulocytes

1981 ◽  
Vol 195 (2) ◽  
pp. 389-397 ◽  
Author(s):  
D A Wiginton ◽  
M S Coleman ◽  
J J Hutton
Keyword(s):  
Molecular Weight ◽  
Adenosine Deaminase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Periodic Acid Schiff ◽  
Apparent Homogeneity

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.

Concanavalin A-horseradish peroxidase bridge staining of alpha-1 glycoproteins separated by isoelectric focusing on polyacrylamide gel.

1976 ◽  
Vol 24 (8) ◽  
pp. 908-914 ◽  
Author(s):  
R C Allen ◽  
S S Spicer ◽  
D Zehr
Keyword(s):  
Horseradish Peroxidase ◽  
Isoelectric Focusing ◽  
Concanavalin A ◽  
Periodic Acid ◽  
Polyacrylamide Gel ◽  
New Method ◽  
Polyacrylamide Gels ◽  
Periodic Acid Schiff ◽  
Coomassie Blue

The Coomassie Blue protein stain and the periodic acid-Schiff stain for glycoproteins are compared to a new method of staining glycoproteins resolved electrophoretically. The method utilizes a Concanavalin A-horseradish peroxidase sequence to visualize selectively glycoproteins with terminal or internal mannose or terminal N-acetylglucosamine. The method applied to characterization of M and Z allele products of alpha-l-antitrypsins separated by isoelectric focusing of polyacrylamide gels slabs have revealed differences in carbohydrate content of various components that were previously undetected.

scholarly journals Platelet membrane studies in the May-Hegglin anomaly

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 279-284 ◽  
Author(s):  
BS Coller ◽  
MH Zarrabi
Keyword(s):  
Sialic Acid ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Glycoprotein ◽  
Galactose Oxidase ◽  
Periodic Acid Schiff ◽  
Giant Platelets ◽  
Total Sialic Acid ◽  
Bernard Soulier Syndrome

Abstract Since studies of the giant platelets in the Bernard-Soulier syndrome have shown decreased electrophoretic mobility, decreased sialic acid, and an abnormality in a membrane glycoprotein, we performed similar studies on the giant platelets from two patients with the May-Hegglin anomaly. The patients' platelet electrophoretic mobilities did not differ from control. Although the total sialic acid contents of the patients' platelets were greater than control when calculated per platelet, they were very similar to control when normalized for differences in platelet volume and surface area. When platelet proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis there were no differences between the glycoproteins of control and patient platelets as judged by the patterns of periodic acid Schiff staining and fluorescein-labeled concanavalin A binding. Similarly, patterns of surface glycoprotein labeling by neuraminidase/galactose oxidase/KB3H4 were identical. We conclude that unlike the giant platelets in the Bernard-Soulier syndrome, those of the May-Hegglin anomaly are not associated with a membrane abnormality detectable by these techniques.

Simplified sodium dodecyl sulfate-polyacrylamide gel electrophoresis of unconcentrated urine with enhanced resolution and detection sensitivity.

1987 ◽  
Vol 33 (10) ◽  
pp. 1886-1887 ◽  
Author(s):  
T Marshall ◽  
K M Williams
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Detection Sensitivity ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Dodecyl Sulfate

Abstract We applied a simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis method to urine. The method, developed for serum protein analysis (Clin Chem 1984;30:475-9), has a high sample throughput and gives excellent resolution with unconcentrated urine. It clearly distinguishes and characterizes proteinuric urine (7.5 microL) by Coomassie Blue staining and gives complex silver-stained patterns with nonproteinuric urine (2 microL). The former is recommended for routine clinical screening, the latter for research purposes.

scholarly journals Specific protein and glycoprotein deficiencies in platelets isolated from two patients with the gray platelet syndrome

Blood ◽  
1982 ◽  
Vol 59 (4) ◽  
pp. 709-718 ◽  
Author(s):  
AT Nurden ◽  
TJ Kunicki ◽  
D Dupuis ◽  
C Soria ◽  
JP Caen
Keyword(s):  
Molecular Weight ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Blue Staining ◽  
Membrane Glycoproteins ◽  
Periodic Acid Schiff ◽  
Platelet Factor ◽  
Sds Page ◽  
Platelet Disorder ◽  
Gray Platelet Syndrome

Abstract The gray platelet syndrome is a rare inherited platelet disorder characterized by the absence of alp ha-granules as observed by electron microscopy. Analysis of the glycoprotein composition of the platelets of 2 such patients by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed decreased or absent staining for carbohydrate of several high molecular weight glycoproteins. The major periodic acid Schiff (PAS) staining membrane glycoproteins were normally detected and were normally labeled with 125I during lactoperoxidase-catalyzed iodination. Analysis of the protein composition of gray platelets by single or two- dimensional SDS-PAGE followed by Coomassie blue staining revealed an apparent absence of GP Ig (thrombospondin), markedly reduced platelet fibrinogen and albumin concentrations, and severely reduced levels of 2 low molecular weight polypeptides exhibiting identical rates of migration on SDS-PAGE as platelet factor 4 and beta-thromboglobulin. SDS-PAGE profiles similar to those of the gray platelets were observed with normal human platelets that had undergone the release reaction induced by thrombin. Analysis of gray platelet proteins by crossed immunoelectrophoresis using a rabbit anti-human platelet antibody preparation and rocket immunoelectrophoresis using monospecific antisera confirmed the above findings and showed additional severe deficiencies of factor VIIIR:Ag and cold-insoluble globulin. In contrast, factor XIII (subunit A), a cytoplasmic protein, was normally detected. Our studies provide further evidence that circulating gray platelets specifically lack, or have markedly decreased concentrations of, the alpha-granule proteins.

scholarly journals Rapid Coomassie Blue Staining and Destaining of Polyacrylamide Gels

BioTechniques ◽  
1996 ◽  
Vol 20 (3) ◽  
pp. 386-388 ◽  
Author(s):  
William Wu ◽  
Michael J. Welsh
Keyword(s):  
Blue Staining ◽  
Polyacrylamide Gels ◽  
Coomassie Blue Staining ◽  
Coomassie Blue
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