Electrophoretic Analysis of Polypeptides of Plasma Membranes from Bovine Pituitary Gland

1974 ◽  
Vol 52 (7) ◽  
pp. 620-630
Author(s):  
André Lemay ◽  
Fernand Labrie
Keyword(s):  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Electrophoretic Pattern ◽  
Apparent Molecular Weight ◽  
Electrophoretic Analysis ◽  
Polyacrylamide Gel ◽  
Protein Components ◽  
Membrane Components

Purified plasma membranes from bovine hypophyseal tissue have been fractionated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under various conditions of pH and acrylamide concentrations. The best separation of protein components is achieved at a concentration of 7.5% acrylamide and at pH 7.1. Under these conditions, the electrophoretic pattern consistently shows 36 protein bands ranging in molecular weights from 250 000 to 15 000. Only one broad band, having an apparent molecular weight of 150 000, stains for glycoproteins by the period acid – Schiff technique. After electrophoresis on a two-dimensional polyacrylamide gel system using disc gels containing urea and Triton X-100 in the first dimension and SDS in the second dimension, approximately 45 different protein components can be identified. Less than 12% of the membrane proteins are solubilized by washing the membranes with 1 M KCl or NH4Cl. Denaturating agents like urea and lithium 3,4-diiodosalycilate solubilize 55–60% of membrane components. Adenohypophyseal plasma membranes show an eleetrophoretic pattern completely different from that obtained with membranes isolated from the intermediate or posterior pituitary lobes.

Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin

Blood ◽  
2001 ◽  
Vol 98 (13) ◽  
pp. 3626-3634 ◽  
Author(s):  
Venke Skibeli ◽  
Gro Nissen-Lie ◽  
Peter Torjesen
Keyword(s):  
Human Serum ◽  
Gel Electrophoresis ◽  
High Performance ◽  
Magnetic Beads ◽  
Direct Method ◽  
Polyacrylamide Gel Electrophoresis ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Two Dimensional Gel Electrophoresis

Abstract Erythropoietin (EPO) from sera obtained from anemic patients was successfully isolated using magnetic beads coated with a human EPO (hEPO)–specific antibody. Human serum EPO emerged as a broad band after sodium dodecyl sulfate–polyacrylamide gel electrophoresis, with an apparent molecular weight slightly smaller than that of recombinant hEPO (rhEPO). The bandwidth corresponded with microheterogeneity because of extensive glycosylation. Two-dimensional gel electrophoresis revealing several different glycoforms confirmed the heterogeneity of circulating hEPO. The immobilized anti-hEPO antibody was capable of binding a representative selection of rhEPO glycoforms. This was shown by comparing normal-phase high-performance liquid chromatography profiles of oligosaccharides released from rhEPO with oligosaccharides released from rhEPO after isolation with hEPO-specific magnetic beads. Charge analysis demonstrated that human serum EPO contained only mono-, di-, and tri-acidic oligosaccharides and lacked the tetra-acidic structures present in the glycans from rhEPO. Determination of charge state after treatment of human serum EPO with Arthrobacter ureafaciens sialidase showed that the acidity of the oligosaccharide structures was caused by sialic acids. The sugar profiles of human serum EPO, describing both neutral and charged sugar, appeared significantly different from the profiles of rhEPO. The detection of glycan structural discrepancies between human serum EPO and rhEPO by sugar profiling may be significant for diagnosing pathologic conditions, maintaining pharmaceutical quality control, and establishing a direct method to detect the misuse of rhEPO in sports.

A Sensitive Peroxidase Staining Immunoblotting Method for Measuring Total Protein S in Human Plasma

1993 ◽  
Vol 69 (04) ◽  
pp. 331-334 ◽  
Author(s):  
Xiangan Li ◽  
Kaoru Hatanaka ◽  
Ling Guo ◽  
Motoo Tsushima ◽  
Yukihiko Kitamura ◽  
...  
Keyword(s):  
Human Plasma ◽  
Total Protein ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Protein S ◽  
Polyacrylamide Gel ◽  
Free Form

SummaryIn plasma, protein S is found in its free form and as a complex with C4b-binding protein. After 125I-protein S was added tonormal human plasma and applied to SDS-8% polyacrylamide gel electrophoresis, the autoradiogram of the gel showed only one single band at free protein S position. Applying this evidence, we have developed a peroxidase staining Western Blotting method to quantitate total protein S in human plasma which consists of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining method (ABC technique). The measurement of protein S by the immunoblotting was reproducible and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected. C4b-binding protein did not affect the measurement of protein S. Compared to other immunoassays, this peroxidase staining immunoblotting method has the advantage of directly estimating the apparent molecular weight of protein of interest, eliminating nonspecific stain and having high sensitivity without using radioisotope.

scholarly journals Cytochrome b-245 from human neutrophils is a glycoprotein

1985 ◽  
Vol 227 (3) ◽  
pp. 783-788 ◽  
Author(s):  
A M Harper ◽  
M F Chaplin ◽  
A W Segal
Keyword(s):  
Cytochrome B ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Human Neutrophils ◽  
Schiff Reagent

Cytochrome b-245 is a glycoprotein. It runs as a broad band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and its apparent Mr varies with the concentration of acrylamide. It stained positively with Schiff reagent and with silver stains after oxidation with periodic acid. It preferentially bound the lectin of Phaseolus vulgaris (type III), and cleavage of carbohydrate with endoglycosidase F resulted in a sharp band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 55000 G.l.c. analysis of carbohydrate showed this to account for about 15% of the Mr and N-acetylglucosamine and galactose to be the major sugars.

scholarly journals Size characteristics of human leucocyte interferon under reducing and non-reducing conditions

1981 ◽  
Vol 193 (3) ◽  
pp. 947-951 ◽  
Author(s):  
I A Braude ◽  
L S Lin ◽  
W E Stewart
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Polyacrylamide Gel ◽  
Lower Molecular Weight ◽  
Reducing Conditions ◽  
Size Heterogeneity

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was used to characterize human leucocyte interferon (HuIFN-alpha) under reducing and non-reducing conditions. Under non-reducing conditions HuIFN-alpha possesses two size forms, but under reducing conditions (r-HuIFN-alpha) only one is observed. The apparent molecular weight of this one form varies with the concentration of 2-mercaptoethanol used. When r-HuIFN-alpha is permitted to reoxidize the bimodal configuration of HuIFN-alpha is restored. The size heterogeneity of native HuIFN-alpha can be eliminated by mild treatment with NaIO4 [HuIFN-alpha/IO4; Stewart II, Lin, Wiranowska-Stewart & Cantell (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4200-4204]. The size of the HuIFN-alpha/IO4 increases after treatment with 2-mercaptoethanol (r-HuIFN-alpha/IO4) and the apparent molecular weight of this component also varies with the concentration of 2-mercaptoethanol used. In the case of r-HuIFN-alpha the single peak observed apparently originates from both the higher- and lower-molecular-weight components.

Isoenzyme and protein patterns of pentose-fermenting yeasts

1987 ◽  
Vol 33 (11) ◽  
pp. 1017-1023 ◽  
Author(s):  
Robert S. Jeng ◽  
Shiyuan Yu ◽  
Morris Wayman
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electrophoretic Pattern ◽  
Pichia Stipitis ◽  
Culture Collection ◽  
Polyacrylamide Gel ◽  
The Other ◽  
Two Dimensional ◽  
Protein Patterns

Soluble proteins were extracted from the vegetative cells of four pentose-fermenting yeasts, Candida shehatae, Pichia stipitis, R-1, and R-2, the R strains being of uncertain taxonomy, while the other two are culture collection yeasts. Isoenzyme patterns, protein patterns, and two-dimensional polypeptide mapping of these four strains were compared by polyacrylamide gel electrophoresis. The two R strains showed great similarity in two-dimensional polypeptide mapping, the pattern of sodium dodecyl sulfate – polyacrylamide gel electrophoresis, isoelectrofocusing, and isoenzymes, and may be one species. Each of the other two yeasts had its own characteristic electrophoretic pattern. The R strains showed the presence of three alcohol dehydrogenase isoenzymes compared with one for the culture collection yeasts, as well as much higher activity of malate dehydrogenase, isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase, which further the formation of pyruvate and ethanol.

scholarly journals Proteins of the kidney microvillus membrane. Identification of subunits after sodium dodecylsullphate/polyacrylamide-gel electrophoresis

1976 ◽  
Vol 159 (2) ◽  
pp. 395-407 ◽  
Author(s):  
A G Booth ◽  
A J Kenny
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Neutral Endopeptidase ◽  
Polyacrylamide Gel ◽  
British Library ◽  
Major Band ◽  
Microvillus Membrane

The proteins of microvilli prepared from pig kidney were analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The typical pattern stained for protein revealed five major bands, four of which also stained for carbohydrate, and about 15 minor bands. For descriptive purposes the bands were designated numerically by their apparent molecular weights (X10(-3). Well-characterized proteins were identified with four of the five major bands. Dipeptidyl peptidase IV, a serine proteinase that may be specifically labelled with di-isopropyl [32P]phosphorofluoridate, was assigned to band 130. Aminopeptidase M was assigned to band 160, though when released from the membrane by a proteinase, this protein comprises three polypeptides each of lower apparent molecular weight than the native enzyme. Neutral endopeptidase can be assigned to band 95 and actin to band 42. The fifth major band (180) is an extrinsic glycoprotein that has not been identified with any microvillus enzyme activity. These four proteins contribute 21% of the microvillus-membrane protein. Kidney microvillus actin was characterized by a variety of properties and was similar to muscle actin. A computer analysis of the gel pattern indicates that it comprises 9.0% of the microvillus protein. Myosin is not present in the microvillus, but another protein associated with band 95, with properties that distinguish it from neutral endopeptidase, was tentatively identified as α-actinin. Alkaline phosphatase was identified as a monomeric polypeptide with an apparent molecular weight of 80000; it is a minor protein of the microvillus and is not discernible as a discrete band in the gel pattern. These and other results permit a model of the organization of the microvillus protein to be suggested. The computer program used has been deposited as Supplementary Publication SUP 50070 (12 pages) at the British Library Lending Division, Boston Spa. Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1976) 153,5.

The mechanism of Fc-mediated interaction of eosinophils with immobilized immune complexes. II. Identification of two membrane proteins, modified by the interaction

1982 ◽  
Vol 56 (1) ◽  
pp. 357-369
Author(s):  
K.J. Thorne ◽  
J. Free ◽  
D. Franks ◽  
R.C. Oliver
Keyword(s):  
Cell Surface ◽  
Heavy Chain ◽  
Plasma Membranes ◽  
Human Peripheral Blood ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Surface Proteins ◽  
Agar Layer ◽  
Coated Surfaces

Human peripheral blood eosinophils attach to and flatten down onto antibody-coated surfaces and subsequently degranulate. An antibody-coated surface was prepared by treating a layer of agar, containing tetanus toxoid antigen and eosinophil chemotactic factor (ECF), with human anti-tetanus immunoglobin. Changes in eosinophil surface proteins during attachment to the antibody-coated agar layer were detected by lactoperoxidase catalysed iodination. Purified eosinophils were pre-treated with unlabelled iodide, lactoperoxidase and hydrogen peroxide to block pre-existing accessible tyrosine residues on the cell surface. They were then allowed to interact with the agar layer, and subsequently treated with lactoperoxidase and 125I-labelled iodide to label newly accessible surface proteins. Separation of the radioactive proteins by sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed that, while incubation of the cells in suspension restored the major proteins to the cell surface, interaction with the antibody-coated agar layer caused the appearance of additional proteins of apparent molecular weight 55K, 30K, 28K and 18K. The 55K, 28K and 18K proteins were greatly reduced when antibody was absent, but the 55K protein was distinguishable from immunoglobulin G (IgG) heavy chain, since it could be detected in low amounts even in the absence of antibody. It was found in purified plasma membranes and it could be separated from IgG heavy chain by iso-electric focusing. The possibility is discussed that this protein is either linked to the receptor for the Fc portion of IgG, or that it is itself the receptor. The 18K protein required both antibody and ECF for maximum expression, but was seen in limited amounts with ECF alone. Possibly it is concerned with an ECF-mediated recognition of IgG. Unlike the 55K protein, it binds concanavalin A. Plasma membranes were prepared from eosinophils by lysis in borate, followed by purification on a glass-bead column. Both the 55K and the 18K proteins were found to be major components of the eosinophil membrane.

Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2018 ◽  
Vol 26 (2) ◽  
pp. 058
Author(s):  
Anna P. Roswiem ◽  
Triayu Septiani
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sds Page

<em>Bahan<strong> </strong>baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE).<strong> </strong>Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi  dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.</em>

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