scholarly journals Monoclonal antibodies to human vitamin K-dependent protein S

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1583-1590 ◽  
Author(s):  
RD Litwiller ◽  
RJ Jenny ◽  
JA Katzmann ◽  
RS Miller ◽  
KG Mann
Keyword(s):  
Monoclonal Antibodies ◽  
Ammonium Sulfate ◽  
High Performance ◽  
Dextran Sulfate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Sds Page ◽  
Dependent Protein ◽  
Hybridoma Technology

Abstract Monoclonal antibodies to human protein S have been prepared using established hybridoma technology. One antibody was isolated that binds protein S only when Ca2+ is present; others bind antigen equally well in the presence or absence of EDTA. Other antibodies display a diminished affinity for protein S in the presence of EDTA. Purified immunoglobulins from cell lines displaying Ca2+ dependence were immobilized and used to purify protein S from fractions obtained by barium precipitation of citrated plasma, ammonium sulfate fractionation, and chromatography on diethylaminoethanol (DEAE)- Sephadex and dextran sulfate agarose. Essentially homogeneous protein S was isolated from the barium-citrate-adsorbed, 35% ammonium-sulfate- soluble proteins using a totally Ca2+-dependent antibody and EDTA elution. Protein S and several substances of higher mol wt were bound directly from plasma by a partially Ca2+-dependent antibody and were eluted partially with EDTA and NaCl and finally with NaSCN. The largest and most abundant of the high mol wt materials is likely protein S- complement C4b-binding protein complex. The immunoaffinity-isolated protein S was found to be indistinguishable from conventionally isolated protein S in terms of activity, sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) mobility, and by high- performance liquid chromatography (HPLC). These studies establish reagents that can probe the structure of protein S and isolate protein S in its free and complexed forms.

scholarly journals Monoclonal antibodies to human vitamin K-dependent protein S

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1583-1590
Author(s):  
RD Litwiller ◽  
RJ Jenny ◽  
JA Katzmann ◽  
RS Miller ◽  
KG Mann
Keyword(s):  
Monoclonal Antibodies ◽  
Ammonium Sulfate ◽  
High Performance ◽  
Dextran Sulfate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Sds Page ◽  
Dependent Protein ◽  
Hybridoma Technology

Monoclonal antibodies to human protein S have been prepared using established hybridoma technology. One antibody was isolated that binds protein S only when Ca2+ is present; others bind antigen equally well in the presence or absence of EDTA. Other antibodies display a diminished affinity for protein S in the presence of EDTA. Purified immunoglobulins from cell lines displaying Ca2+ dependence were immobilized and used to purify protein S from fractions obtained by barium precipitation of citrated plasma, ammonium sulfate fractionation, and chromatography on diethylaminoethanol (DEAE)- Sephadex and dextran sulfate agarose. Essentially homogeneous protein S was isolated from the barium-citrate-adsorbed, 35% ammonium-sulfate- soluble proteins using a totally Ca2+-dependent antibody and EDTA elution. Protein S and several substances of higher mol wt were bound directly from plasma by a partially Ca2+-dependent antibody and were eluted partially with EDTA and NaCl and finally with NaSCN. The largest and most abundant of the high mol wt materials is likely protein S- complement C4b-binding protein complex. The immunoaffinity-isolated protein S was found to be indistinguishable from conventionally isolated protein S in terms of activity, sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) mobility, and by high- performance liquid chromatography (HPLC). These studies establish reagents that can probe the structure of protein S and isolate protein S in its free and complexed forms.

Study on Degradation of Radix Isatidis Polypeptide in Artificial Gastrointestinal Environment

2014 ◽  
Vol 989-994 ◽  
pp. 1020-1024
Author(s):  
Nan Nan ◽  
Xi Jing Liu
Keyword(s):  
Gel Electrophoresis ◽  
Free Amino ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Radix Isatidis ◽  
Sds Page ◽  
Electrophoresis Method ◽  
Amino Acid Levels ◽  
Different Molecular Weight

Radix Isatidis is a traditional Chinese medicine for treatment of influenza and inflammation in China. In this paper, in order to study the degradation situation of Radix Isatidis polypeptide in artificial gastrointestinal environment, the SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) method was used to detect the degradation of Radix Isatidis polypeptide in artificial intestinal juice and gastric juice, and it showed that Radix Isatidis peptides could be degradated to different degrees. HPLC (High Performance Liquid Chromatography) was used to determine the change of peptides degradation, and it indicated that free amino acid levels did not change significantly. The result after degradation was also detected by BCA method, and it showed that there were still a large number of polypeptides in the liquid. From this experiment we can come to this conclusion that Radix Isatidis polypeptides in artificial gastrointestinal juice mostly degraded into a series of different molecular weight peptides.

scholarly journals Production and characterisation of monoclonal antibodies against immunoglobulins of Cirrhinus mrigala (Hamilton 1822)

2020 ◽  
Vol 67 (2) ◽  
Author(s):  
Snatashree Mohanty ◽  
M. Makesh ◽  
K. V. Rajendran ◽  
P. P. Suresh Babu ◽  
Deepika Anand ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Catla Catla ◽  
Cirrhinus Mrigala ◽  
Indian Major Carps ◽  
Sds Page ◽  
Igg1 Isotype ◽  
Major Carps

Serum immunoglobulins (Ig) of mrigal Cirrhinus mrigala (Hamilton 1822) immunised with bovine serum albumin (BSA), were purified by affinity chromatography using BSA-CL agarose column. The purified mrigal Ig (m-Ig) was characterised under reducing condition by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) which revealed two bands of 85 and 26 kDa corresponding to heavy and light chain, respectively. Following fusion of splenocytes from Balb/c mice immunised with purified m-Ig with myeloma cells, three hybridomas showing reactivity with m-Ig were cloned by limiting dilution. The monoclonal antibodies (MAbs) generated by these clones were designated as 3B2-E12, 3B2-F9 and 4C3-B2 and characterised by western blotting and isotyping. Western blot analysis of the supernatant from the three clones with purified m-Ig indicated that, all the three MAbs were specific to heavy chain. Isotyping revealed that 3B2-E12 MAb was of IgG1 isotype whereas the other two MAbs were of IgG2a isotype. Cross reactivity of anti-mrigal Ig MAb (3B2-E12) was observed with serum Ig of Catla catla and Labeo rohita indicating semi-conserved nature of Ig in Indian major carps.

scholarly journals Extraction, Characterization and Antioxidant Activity in vitro of Proteins from Semen Allii Fistulosi

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3235
Author(s):  
Min Zuo ◽  
Xiao-xiao Liu ◽  
Di Liu ◽  
Hang-yun Zhao ◽  
Lu-lu Xuan ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
High Performance ◽  
Orthogonal Design ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Reducing Power ◽  
Raw Material ◽  
Extraction Solvent ◽  
Sds Page

Semen Allii Fistulosi (PSAF) is the seed of Allium fistulosum L. of the Liliaceae family. The purpose of this study was to extract, characterize, and evaluate the antioxidant activity in vitro of proteins. Using single factor and orthogonal design, the optimum conditions of extraction were determined to be as follows: extraction time 150 min, pH 8.5, temperature 60 °C, and ratio (v/w, mL/g) of extraction solvent to raw material 35. The isoelectric point of the pH was determined to be about 4.4 and 10.2, by measuring the protein content of PSAF solutions at different pH values. The amino acid composition of PSAF was determined by high performance liquid chromatography (HPLC), and the results suggested that the species of amino acids contained in the PSAF was complete. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) analysis showed the molecular weight was mainly between 40 and 55 kDa, and Fourier-transform infrared spectroscopy (FTIR) characterized prevalent protein absorption peaks. PSAF exhibited potent scavenging activities against DPPH assays, via targeting of hydroxyl and superoxide radicals, while chelating Fe2+ activity and demonstrating weak reducing power. This work revealed that PSAF possessed potential antioxidant activity in vitro, suggesting potential for use of PSAF as a natural antioxidant.

scholarly journals Unique role of the complement receptor CR1 in the degradation of C3b associated with immune complexes.

1982 ◽  
Vol 156 (6) ◽  
pp. 1739-1754 ◽  
Author(s):  
M E Medof ◽  
K Iida ◽  
C Mold ◽  
V Nussenzweig
Keyword(s):  
Monoclonal Antibodies ◽  
Immune Complexes ◽  
Solid Phase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alpha Chain ◽  
Sds Page ◽  
High Concentrations ◽  
Polypeptide Chains

The main finding of this paper is that CR1, the membrane receptor for C3b and C4b, together with C3b/C4b-inactivator (I), degrades C3b bound to immune complexes (C3b*). Two fragments are generated: C3c, which is released from the immune complexes, and C3d*. The C3c fragment released from the cell intermediate EAC1423b prepared with 125I-C3 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. It has a 135,000 mol wt and contains disulfide bonded labeled polypeptide chains of 75,000 and 31,000 mol wt, which presumably represent the beta and a fragment of the alpha-chain of C3b*. Silver staining of the SDS-PAGE gels revealed other C3-derived bands with 39-42,000 mol wt. Human erythrocytes + I also cleave C3b* into C3c and C3d*. The activity of the erythrocytes is CR1 mediated because it can be totally inhibited by monoclonal antibodies to CR1. In contrast with these results, I together with the serum protein beta 1H (H) transform EAC1423b into hemolytically inactive EAC1423bi and cleave the alpha' chain of C3b* into fragments of 70,000 and 40,000 mol wt. Small amounts of C3c are also released at relatively high concentrations of H. On a molar basis, the efficiency of CR1 in the generation of C3c and C3d is 10(4)-10(5) greater than H. An additional observation was that C3c could be released by treating EAC1423bi with CR1 + I and that this reaction was also inhibited by monoclonal antibodies to CR1. Therefore, it is likely that CR1 has binding affinity for iC3b and that the degradation of C3b* proceeds as follows: C3b (formula, see text) C3c + C3d*. Taken together, our findings argue that the processing of C3b* in vivo occurs in solid phase, that is, on the surface of cells bearing CR1.

Comparison of plasma membrane FABP and mitochondrial isoform of aspartate aminotransferase from rat liver

1993 ◽  
Vol 265 (5) ◽  
pp. G894-G902 ◽  
Author(s):  
D. D. Stump ◽  
S. L. Zhou ◽  
P. D. Berk
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Amino Acid ◽  
Rat Liver ◽  
Aspartate Aminotransferase ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fatty Acid Binding ◽  
Sds Page

A relationship between plasma membrane fatty acid binding protein (FABPpm), a putative membrane transporter for long-chain fatty acids, and the mitochondrial isoform of aspartate aminotransferase (m-AspAT) has been reported. Accordingly, we have compared the chemical and immunological properties of rat liver m-AspAT with those of rat liver FABPpm isolated by two procedures: 1) detergent solubilization of the membranes followed by purification via fatty acid affinity chromatography (FABP-1) or 2) salt extraction of the membranes and subsequent purification by high-performance liquid chromatography (HPLC; FABP-2). Comparison of the three protein preparations revealed no differences with respect to NH2-terminal amino acid sequence, amino acid composition, peptides from tryptic digests, AspAT enzymatic activity, isoelectric point, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), retention on five different HPLC columns, and immunoprecipitation and immunoblotting of SDS-PAGE separated proteins with polyclonal antisera. Examination of the proteins by nondenaturing PAGE showed a consistent second band in FABP-1 and FABP-2 not always present in m-AspAT. However, whenever present, this band was immunoreactive with antibodies to both m-AspAT and FABP-1. Hence, FABP-1 and FABP-2 are indistinguishable from one another. They are also at least closely related, if not identical, to m-AspAT.

scholarly journals Role of plasmin in the degradation of the stroma-derived fibrin in human ovarian carcinoma

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1673-1678 ◽  
Author(s):  
O Wilhelm ◽  
R Hafter ◽  
A Henschen ◽  
M Schmitt ◽  
H Graeff
Keyword(s):  
Ovarian Cancer ◽  
High Performance ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fibrin Degradation Products ◽  
Sds Page ◽  
Human Ovarian Carcinoma

Abstract The aim of this study was to evaluate the type of enzymes involved in tumor-associated fibrinolysis of the stroma component fibrin in ovarian cancer patients. For this purpose, the high-molecular-mass fibrin degradation products (HMM-XDP) were isolated from malignant ascitic fluid by protamine sulfate precipitation and further purified by gel filtration and acid precipitation. After reduction with 2- mercaptoethanol, the peptide chain components were separated by reverse- phase high-performance liquid chromatography (RP-HPLC). The nature of these components was elucidated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequence analysis and compared with fibrin-derived fragments formed in vitro. The results indicate that plasmin is the essential protease involved in the degradation of the stroma-derived fibrin portion found in ovarian cancer ascites.

scholarly journals Isolation and Identification of Protein Spike (Protein-S) in Field Isolates of Infectious Bronchitis Virus

2021 ◽  
Vol 4 (1) ◽  
pp. 104
Author(s):  
Jola Rahmahani ◽  
Dewanggi Kristi Sasmi Pradhita ◽  
Nanik Sianita Widjaja ◽  
Suwarno Suwarno
Keyword(s):  
Infectious Bronchitis Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Field Isolate ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Spike Protein ◽  
Structural Protein ◽  
Isolation And Identification ◽  
Infectious Bronchitis ◽  
Sds Page

Infectious Bronchitis (IB) is disease causes great economic impact across nations. Spike protein (protein-S) is one of structural protein of Infectious Bronchitis Virus (IBV) located on enveloped of the virion. Molecular characterization of IBV could be conducted use Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to reveal the protein weight then confirmed through Immuno-Blotting assay. These methods were conducted to know the molecule weight of field isolate compared to seed vaccine. The results of this study indicate the weight of protein-S of field isolate I-147/11 were 168.21 kDa which was same to protein-S of seed vaccine Massachusset.

scholarly journals An apparently higher molecular weight gamma-chain variant in a new congenital abnormal fibrinogen Tochigi characterized by the replacement of gamma arginine-275 by cysteine

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 480-487
Author(s):  
N Yoshida ◽  
K Ota ◽  
M Moroi ◽  
M Matsuda
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Positive Staining ◽  
Gamma Chain ◽  
Peptide Peak ◽  
Sds Page ◽  
Fibrin Monomers ◽  
Chain Variant

A gamma-chain variant with an apparently higher molecular weight than the normal gamma-chain was detected in a new congenital abnormal fibrinogen with impaired polymerization of the fibrin monomer and with normal release of fibrinopeptides A and B in a 51-year-old male. Purified fibrinogen analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the reduced condition in the system of Laemmli contained two protein bands in the gamma-chain region (molecular weight, 50,500 as compared with 50,000 for the normal), both with normal crosslinking ability. The presence of two types of gamma- chains was more clearly detected when reduced and carboxymethylated fibrinogen was analyzed by SDS-PAGE or when reduced fragment D2 was analyzed on SDS-PAGE followed by Western blotting, and identified by positive staining for anti gamma-chain monoclonal antibody. Cyanogen bromide- or lysylendopeptidase-cleavage of purified gamma-chains analyzed on reverse-phase high performance liquid chromatography showed the decrease of one peptide compared with the normal and the appearance of an abnormal peptide peak. Amino acid sequence analysis demonstrated that the gamma arginine-275 of gamma-chain variant was replaced by a cysteine. These data suggest that some regions or conformations containing gamma 275 will affect the polymerization of fibrin monomers. The propositus' two daughters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Tochigi, and the gamma-chain variant as gamma Tochigi.

scholarly journals Isolation of an abnormal protein C molecule from the plasma of a patient with thrombotic diathesis

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 940-946
Author(s):  
EM Faioni ◽  
CT Esmon ◽  
NL Esmon ◽  
PM Mannucci
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Protein C ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Activated Protein C ◽  
Protein S ◽  
Abnormal Protein ◽  
Sds Page ◽  
Before And After

Protein C has been purified from the plasma of a patient with thrombotic diathesis. Both before and after isolation, the protein showed reduced capacity to hydrolyze synthetic substrates and to anticoagulate plasma. Proteolysis with the soluble thrombin- thrombomodulin complex proceeded normally and to completion as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Approximately one-third of the protein is functional, indicating a heterozygous defect. Indirect studies suggest that the abnormal component can bind to protein S and phospholipids. Both forms of activated protein C can also incorporate radiolabeled diisopropylfluorophosphate.

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