scholarly journals Characterization of Immunochemically Nonreactive Urinary Albumin

2004 ◽  
Vol 50 (12) ◽  
pp. 2286-2291 ◽  
Author(s):  
Tanya M Osicka ◽  
Wayne D Comper
Keyword(s):  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Clinical Importance ◽  
Urinary Albumin ◽  
Albumin Concentration ◽  
Urinary Proteins ◽  
Native Page ◽  
Sds Page ◽  
Urinary Albumin Concentration

Abstract Background: Conventional immunoassays underestimate the urinary albumin concentration because intact albumin in urine exists in two forms, immunoreactive and immunochemically nonreactive. Methods: Urinary albumin concentration measured by HPLC (which measures total albumin, i.e., the sum of immunoreactive albumin + immunochemically nonreactive albumin) or RIA was compared with densitometric analysis of albumin bands in diabetic urine samples separated by either native polyacrylamide gel electrophoresis (PAGE) or reducing sodium dodecyl sulfate (SDS)-PAGE. Immunochemically nonreactive albumin was also isolated from diabetic urine (relative amount detected, 70–80% of the expected) and was tested for contamination by common urinary proteins by native PAGE, ELISA, and capillary electrophoresis. Results: Urinary albumin concentrations measured by native PAGE and HPLC were better correlated (r2 = 0.83) than concentrations measured by native PAGE and RIA (r2 = 0.62) because under native conditions both native PAGE and HPLC detect total albumin and not only the immunoreactive albumin alone that is measured by RIA. Urinary albumin concentrations measured by reducing SDS-PAGE and RIA were better correlated (r2 = 0.84) than concentrations measured by reducing SDS-PAGE and HPLC (r2 = 0.65) because under reducing conditions immunochemically nonreactive albumin is unstable and fragments into many smaller peptides. The partially purified preparation was found to contain <1% contamination by common urinary proteins and is stable to freezing and frequent freeze/thaw cycles. Conclusions: The results are consistent with the interpretation that immunochemically nonreactive albumin has a limited number of polypeptide chain scissions and is held together by noncovalent intrachain bonding and disulfide bonds. Detection of this molecule is likely to be of clinical importance in diagnosing kidney disease as well as cardiovascular disease.

scholarly journals Bovine Herpesvirus 1 Glycoprotein M Forms a Disulfide-Linked Heterodimer with the UL49.5 Protein

1998 ◽  
Vol 72 (4) ◽  
pp. 3029-3036 ◽  
Author(s):  
S. X. Wu ◽  
X. P. Zhu ◽  
G. J. Letchworth
Keyword(s):  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Herpesvirus ◽  
In Vitro Translation ◽  
Bovine Herpesvirus 1 ◽  
Western Blots ◽  
Sds Page ◽  
Herpesvirus 1

ABSTRACT Nine glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, and gL) have been identified in bovine herpesvirus 1 (BHV-1). gM has been identified in many other alpha-, beta-, and gammaherpesviruses, in which it appears to play a role in membrane penetration and cell-to-cell fusion. We sought to express BHV-1 open reading frame UL10, which encodes gM, and specifically identify the glycoprotein. We corrected a frameshift error in the published sequence and used the corrected sequence to design coterminal peptides from the C terminus. These were expressed as glutathione S-transferase fusion proteins inEscherichia coli. The fusion protein containing the 63 C-terminal amino acids from the corrected gM sequence engendered antibodies that immunoprecipitated a 30-kDa protein from in vitro translation reactions programmed with the UL10 gene. Proteins immunoprecipitated by this antibody from virus-infected cells ran at 36 and 43 kDa in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 43 and 48 kDa in nonreducing SDS-PAGE. Only the larger of the pair was present in virions. A 7-kDa protein was released from gM by reducing agents. The 7-kDa protein was not recognized in Western blots probed with the anti-gM antibody but reacted specifically with antibodies prepared against BHV-1 UL49.5, previously reported to be a 9-kDa protein associated with an unidentified 39-kDa protein (X. Liang, B. Chow, C. Raggo, and L. A. Babiuk, J. Virol. 70:1448–1454, 1996). This is the first report of a small protein covalently bound to any herpesvirus gM. Similar patterns of hydrophobic domains and cysteines in all known gM and UL49.5 homologs suggest that these two proteins may be linked by disulfide bonds in all herpesviruses.

scholarly journals Migration Behavior of Immunoglobulin Y in Native Page Electrophoresis

2019 ◽  
Vol 2 (1) ◽  
pp. 50-56
Author(s):  
Umul Karimah
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Protein Separation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Migration Behavior ◽  
Immunoglobulin Y ◽  
Native Page ◽  
Nonreducing Condition ◽  
Sds Page

ABSTRACT Protein separation with electrophoresis in native state (native Polyacrylamide Gel Electrophoresis, native PAGE) is rarely applied in laboratory which results in limited scientific report. Native PAGE result can be used in further assay analysis or shows important protein-protein interaction. This study aimed to investigate immunoglobulin Y(IgY) behavior in separation using native PAGE. IgY protein is used since many properties of IgY are already known. IgY was isolated from follicles yolk and examined using clear native PAGE (CN-PAGE) and Sodium Dodecyl Sulphate (SDS) PAGE, both in reducing and nonreducing condition. CN-PAGE electropherogram of IgY showed streaked band from ~409-1048 kDa indicating IgY aggregation. Whereas SDS-PAGE in reducing condition showed normal result, ~68 kDa and ~18,7 kDa protein bands correlated with heavy and light chain of IgY respectively. Moreover SDS PAGE in nonreducing condition also resulted in normal IgY band sized ~185 kDa. Dilution and disaggregation using sodium dodecyl sulphate (SDS), sodium deoxycholate (SDC), sarcosyl, urea, and glycerol did not affect the migration behavior. We concluded that IgY is not in aggregate form. Migration behavior of IgY in CN-PAGE which was slow, produced streaked band, and appeared to have very high molecular weight is possibly due to low electronegativity (pI = 6,6±0,9) in CN-PAGE condition (buffer pH 8,3) and planar conformation of IgY. Keywords: electrophoresis, immunoglobulin Y, native PAGE.

scholarly journals ANALYSIS OF PROTEIN CONCENTRATE FROM KAHAI (CARYODENDRON ORINOCENSE KARST) SEEDS CULTIVATED IN AMAZONIA OF ECUADOR

2018 ◽  
Vol 11 (6) ◽  
pp. 369
Author(s):  
Greffa J ◽  
Vilcacundo E ◽  
Altuna J ◽  
Carrillo W
Keyword(s):  
Extraction Method ◽  
Colorimetric Assay ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heat Extraction ◽  
Precipitation Method ◽  
Protein Concentrate ◽  
Native Page ◽  
Molecular Weights ◽  
Sds Page

Objective: The aim of this study was to obtain Kahai protein concentrate from Caryodendron orinocense Karst cultivated in the Amazonic region of Ecuador, to characterize its proteins using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native electrophoresis methods, and to determine its content of total polyphenols.Methods: Kahai seeds (C. orinocense Karst) were utilized to obtain Kahai protein concentrate at pH 3.0, pH 4.0, pH 5.0, and pH 6.0 using the isoelectric precipitation method. The proteins were characterized using the SDS-PAGE and native-PAGE electrophoresis methods. Total polyphenols were determined using the colorimetric assay method.Results: The best treatment to obtain Kahai protein concentrate was at pH 5.0 with a 21.91% yield using the cold extraction method. The best treatment was at pH 6.0 with a 11.07% yield using the heat extraction method. Kahai concentrates presented a complex profile of proteins with molecular weights between 14 and 97 kDa. It was possible to obtain at the same time polyphenols at pH 5.0 with a value of 1028.58 mg gallic acid equivalents/100 g protein of sample.Conclusion: This study suggests that Kahai protein concentrates possess a high content of proteins and polyphenols that can be used to elaborate functional foods.

Differences between cattle and buffalo in the water-soluble proteins of the Longissimus muscle as shown by electrophoretic techniques

2020 ◽  
Vol 60 (14) ◽  
pp. 1759
Author(s):  
Rafael S. B. Pinheiro ◽  
Paulo R. R. Ramos ◽  
Roberto de O. Roça ◽  
Leilson R. Bezerra ◽  
Caroline L. Francisco ◽  
...  
Keyword(s):  
Animal Species ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Water Soluble ◽  
Longissimus Dorsi ◽  
Muscle Proteins ◽  
Food Fraud ◽  
Native Page ◽  
Buffalo Meat ◽  
Sds Page

Context Fraudulent information about food is an old and widespread problem, particularly regarding products with high economic value, such as meat and meat products. The motivation for food fraud is economic, but it can have serious impacts on public health, thus creating a food security problem. Approximately 90% of buffalo meat is marketed as beef in various regions where the consumption of buffalo meat is considered unusual. Aims To determine the electrophoretic profile of the raw Longissimus dorsi of cattle and buffalo species and to test the hypothesis that electrophoresis techniques can be used to distinguish meat from cattle from buffalo meat. Methods Fourteen 10-g samples of Longissimus dorsi (12th and 13th rib) tissue were taken from each animal of both species after slaughter. The meat of each species was analysed by native polyacrylamide gel electrophoresis (NATIVE PAGE) and by denaturing and non-denaturing sodium dodecyl sulfate (SDS)–PAGE. Differences (P < 0.05) were observed between water-soluble cattle and buffalo muscle proteins in both NATIVE PAGE (relative mobilities and percentages of protein bands) and non-denaturing and denaturing SDS–PAGE (molecular weights in kDa and optical density index). Key results With the NATIVE PAGE technique, 10 protein bands were observed in the gel, and three of these bands exhibited differences between species (P ≤ 0.05). The non-denaturing and denaturing SDS–PAGE techniques yielded significantly different protein bands in the gel. The electrophoretic profiles of some cattle and buffalo muscle proteins are distinct; therefore, raw meat flesh samples of these animal species can be distinguished using these electrophoresis techniques. Conclusions Each of the three electrophoresis techniques used can distinguish meat from different animal species; however, when there is doubt about the animal species, the use of more than one electrophoretic technique is recommended, so as to obtain more reliable results. Implications The use of electrophoresis techniques to differentiate cattle and buffalo meat is promising. This technique could be used in cases of suspected food fraud, such as the replacement of beef with buffalo or vice versa, with reliable results that will be accepted by supervisory bodies.

Mechanism Of The Inactivation Of Trypsin By Antithrombin III

1981 ◽  
Author(s):  
A Danielsson ◽  
I Björk
Keyword(s):  
Disulfide Bonds ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
General Mechanism ◽  
Reducing Conditions ◽  
Sds Page ◽  
Activity Measurements ◽  
General Aspects ◽  
Modified Forms

The reaction between bovine antithrombin III (AT) and bovine trypsin was'studied and compared to the inactivation of thrombin by the inhibitor in order to elucidate general aspects of the mechanism of AT action. AT and trypsin formed inactive 1:1 complexes, as determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/ PAGE) and trypsin activity measurements. In the absence of heparin, the reaction was about 100-fold faster than the AT-thrombin reaction. The reaction rate increased when AT was preincubated with heparin before trypsin was added. Purified AT-trypsin complex dissociated at pH 10 into free enzyme and two proteolytically modified forms of AT, while no intact AT appeared. In SDS/PAGE, the two modified inhibitors gave bands corresponding to apparent molecular weights of 52000 and 48000 under reducing conditions, while both forms co-migrated with intact AT (mol wt 56000) under non-reducing conditions. This indicates that each of the two modified forms of AT had been cleaved into two or more chains held together by disulfide bonds. Under reducing conditions, the larger of the modified AT chains co-migrated grated with the large chain of thrombin-modified AT, i.e. the form of AT which dissociates from the AT-thrombin complex and which is cleaved at the reactive Arg-Ser bond of the inhibitor. Control experiments showed that the smaller of the two chains was formed by tryptic cleavage of the larger chain. Antisera specific for thrombin-modified AT reacted with purified AT-trypsin complex, demonstrating that the inhibitor was present in the complex in a form immunologically identical to thrombin-modified AT. An analogous finding has been reported earlier for the anti thrombin- thrombin complex. Together, these results suggest the same general mechanism for inhibition of trypsin and thrombin by AT.

Glutathione synthetase from the fission yeast. Purification and its unique heteromeric subunit structure

1993 ◽  
Vol 71 (9-10) ◽  
pp. 447-453 ◽  
Author(s):  
Chiaki W. Nakagawa ◽  
Norihiro Mutoh ◽  
Yukimasa Hayashi
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Fission Yeast ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Glutathione Synthetase ◽  
Subunit Structure ◽  
Wild Type ◽  
Native Page ◽  
Sds Page

Glutathione (GSH) synthetase (EC 6.3.2.3) was purified from the fission yeast Schizosaccharomyces pombe L972h− and from the GSH synthetase deficient mutant MN101/pYS41, which harbors a plasmid containing the GSH synthetase gene of the fission yeast. GSH synthetase is expressed at 10 times higher the amount in MN101/pYS41 than in wild-type L972 h−. The purified enzyme gave a single band on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (native PAGE). The molecular weight of this enzyme was determined to be 1.2 × 105 by Sepharose CL-6B gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that this enzyme was composed of two kinds of subunits, A (Mr = 33 × 103) and B (Mr = 26 × 103), and existed as a heterotetramer (A2B2). The enzyme purified from the wild-type fission yeast, which did not harbor the plasmid, showed the same electrophoretic mobilities on both native PAGE and SDS–PAGE and similar catalytic properties under standard conditions. This enzyme is most active at 45 °C and pH 8.0–8.5 with 20 mM Mg2+ + 10 mM ATP and 50 mM K+. The strict requirement for the monovalent cation is rather specific for the enzymes from yeasts. The presence of sugar components in the enzyme is also observed, similar to that in the rat kidney enzyme.Key words: Schizosaccharomyces pombe, glutathione synthetase, heteromeric subunit structure.

Analysis of Proteinuria Using a Commercial System for Automated Electrophoresis and Isoelectric Focusing

1988 ◽  
Vol 25 (3) ◽  
pp. 319-324 ◽  
Author(s):  
P J Jackson ◽  
C J Sampson ◽  
E H Cooper ◽  
D Heney ◽  
J T Brocklebank
Keyword(s):  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tubular Dysfunction ◽  
Urinary Proteins ◽  
Sds Page ◽  
Glomerular Damage ◽  
Bence Jones Proteins

We describe an investigation of proteinuria using Pharmacia PhastSystem™ electrophoresis apparatus. The analysis of urinary proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of unconcentrated urine followed by silver staining took about 2 h and could clearly demonstrate tubular dysfunction or glomerular damage in urines with a negative or only trace-positive dip-stick test for protein. In addition, we show the identification of urinary proteins by immunoblotting from SDS-PAGE gels and the characterisation of Bence-Jones proteins by isoelectric focusing (IEF) and immunoblotting.

scholarly journals Coupling native page/activity-staining with SDS-PAGE/immunodetection for the analysis of glutamine synthetase isoforms in spinach

2011 ◽  
Vol 63 (4) ◽  
pp. 965-969 ◽  
Author(s):  
M. Dragicevic ◽  
Vanja Tanackovic ◽  
Danijela Misic ◽  
Tijana Cvetic ◽  
Sladjana Todorovic ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Posttranslational Modifications ◽  
Spinacia Oleracea ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Methionine Sulfoximine ◽  
Activity Detection ◽  
Native Page ◽  
Sds Page ◽  
Native Polyacrylamide Gel Electrophoresis

Glutamine synthetase (GS) is a key nitrogen-assimilating enzyme in plants and a target for the broad-spectrum herbicide glufosinate. Understanding its kinetic and structural properties is of major agricultural importance. Spinach (Spinacia oleracea) is classified as a plant expressing only chloroplastic GS activity. We have analyzed soluble proteins in the spinach by coupling native polyacrylamide gel electrophoresis (PAGE)-activity detection, based on phosphate precipitation, with SDS-PAGE/immunoblotting. One cytosolic (GS1) isoform from the roots and two chloroplastic (GS2) isoforms expressed in leaves were resolved by native PAGE. The identity of the obtained bands was established by the application of GS-specific inhibitors, L-methionine sulfoximine and glufosinate. Examination by sodium dodecyl sulfate (SDS)-PAGE/ Western analysis with anti-GS antibodies, confirmed the identity of the active bands and revealed that both chloroplastic isoforms are composed of 44 kDa subunits, while the cytosolic isoform consists of 40 kDa subunits. The presence of more GS2 isozymes than encoded in the spinach genome is discussed in terms of posttranslational modifications.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

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