One-step purification of chicken growth hormone from a crude pituitary extract by use of a monoclonal immunoadsorbent

1988 ◽  
Vol 118 (3) ◽  
pp. 381-387 ◽  
Author(s):  
L. R. Berghman ◽  
J. van Beeumen ◽  
E. Decuypere ◽  
E. R. Kühn ◽  
F. Vandesande
Keyword(s):  
Monoclonal Antibodies ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Reversed Phase ◽  
Molecular Forms ◽  
Assay Method ◽  
One Step ◽  
Purification Protocol

ABSTRACT The immunization of mice with an affinity-purified glycoprotein preparation from chicken pituitary tissue yielded several monoclonal antibodies towards the recently described glycosylated variant of chicken GH. As all these antibodies recognize the classical (non-glycosylated) GH molecule equally well, they provide a suitable tool for the development of both a specific immunoadsorbent and an assay method. This paper deals with the surprising purification power of the immunoadsorbent that was produced with one of the monoclonal antibodies. The resulting preparation was more than 99% pure as assessed by reversed phase high-performance liquid chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, so that no further purification steps were needed before the determination of the amino acid sequence of the material. The efficiency of the purification protocol as determined by a homologous, monoclonal antibody-based radioimmunoassay was virtually absolute. Moreover, the affinity-purified GH preparation was a mixture representing the multiple molecular forms of pituitary chicken GH, including both oligomeres and glycosylated GH. The purified preparations were finally used to demonstrate the hepatic 5′-monodeiodinase-stimulating activity of GH in the chicken embryo (results not shown), in order to prove that the biological activity of the molecule had not been damaged by elution from the immunoadsorbent. J. Endocr. (1988) 118, 381–387

Purification of goats' milk casein by reversed-phase high-performance liquid chromatography and identification of αs1-casein

1987 ◽  
Vol 54 (3) ◽  
pp. 361-367 ◽  
Author(s):  
Jan Mikkelsen ◽  
Peter Højrup ◽  
Jens Knudsen
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Reversed Phase ◽  
Terminal Sequence ◽  
Phase Gradient ◽  
Milk Casein

SummaryGoats’ milk caseins were separated into four components in a single run using reversed-phase gradient high-performance liquid chromatography. The purity of the isolated components was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis, amino acid analysis and determination of the N-terminal residue. By a comparison with previously published results for goats’ milk caseins the four peaks were identified as κ-, αs1-, αs2- and β-casein. In order to confirm the existence of αsl-casein in goats’ milk, this component was sequenced for 44 steps, revealing a sequence homologous to bovine αsl-casein and almost identical to the N-terminal sequence previously published by Boulanger et al. (1984).

scholarly journals Reutilization of Food Waste: One-Step Extration, Purification and Characterization of Ovalbumin from Salted Egg White by Aqueous Two-Phase Flotation

Foods ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 286 ◽  
Author(s):  
Bin Jiang ◽  
Jiaxin Na ◽  
Lele Wang ◽  
Dongmei Li ◽  
Chunhong Liu ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Flow Rate ◽  
High Performance ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Egg White ◽  
Two Phase ◽  
One Step ◽  
Egg Yolks

For the purpose of reducing pollution and the reutilization of salted egg whites, which are byproducts of the manufacturing process of salted egg yolks and normally treated as waste, an aqueous two-phase flotation (ATPF) composed of polyethylene glycols (PEG 1000) and (NH4)2SO4 was applied to develop a simple, inexpensive and efficient process for the separation of ovalbumin (OVA) from salted egg whites. The effects of the concentration of PEG, the concentration of (NH4)2SO4, the flow rate and the flotation time on the flotation efficiency (Y) and purity (P) of OVA were investigated. A response surface method (RSM) experiment was carried out on the basis of a single-factor experiment. An efficient separation was achieved using ATPF containing 5 mL of 80% PEG 1000 (w/w), 28 mL of 28% (NH4)2SO4 (w/w), 35 mL/min of the flow rate and 30 min of the flotation time, while 2 mL of the salted egg white solution (salted eggs white (v): water (v) = 1:4) was loaded. Under the optimal conditions, Y and P of OVA could reach 82.15 ± 0.24% and 92.98 ± 0.68%, respectively. The purified OVA was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reverse phase high-performance liquid chromatography (RP-HPLC), liquid chromatography-nano electrospray ionisation mass spectrometry (Nano LC-ESI-MS/MS), ultraviolet spectrum (UV), fluorescence spectrum (FL) and fourier transform infrared spectroscopy (FT-IR). The results indicated that the purity of OVA obtained by ATPF was satisfactory and there was no obvious difference in the structure of the OVA separated by ATPF and the standard. The results of the functional properties revealed no significant differences between OVA obtained by ATPF and the standard in oil binding capacity, viscosity, emulsibility and foam capacity.

scholarly journals Isolation and analysis of human parathyrin in parathyroid tissue and plasma Use of reversed-phase liquid chromatography

1981 ◽  
Vol 197 (2) ◽  
pp. 391-400 ◽  
Author(s):  
H P J Bennett ◽  
S Solomon ◽  
D Goltzman
Keyword(s):  
Liquid Chromatography ◽  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Parathyroid Tissue ◽  
Reversed Phase ◽  
Resolving Power ◽  
Molecular Forms ◽  
Reversed Phase Liquid Chromatography ◽  
Phase Liquid

Reversed-phase liquid chromatography techniques have been used to extract and purify human parathyrin from parathyroid adenomas and to analyse the circulating forms of human parathyrin in plasma. Both the supernatant from tissue homogenates, and plasma were extracted with octadecylsilyl-silica (ODS-silica) in a batch procedure. Extracts were subjected to reversed-phase high-pressure liquid chromatography (h.p.l.c.) employing solvent systems composed of aqueous acetonitrile containing trifluoroacetic acid or heptafluorobutyric acid as hydrophobic ion-pairing reagents. The volatile solvents facilitated the radioimmunoassay, bioassay in vitro and amino acid analysis of column fractions and permitted monitoring for u.v. absorbance at 210nm. Isolated glandular parathyrin was found to be homogeneous by sodium dodecyl sulphate/urea/polyacrylamide-gel electrophoresis, to have an amino acid composition conforming to that of human parathyrin-(1-84)-tetraoctacontapeptide and to be bioactive in both renal adenylate cyclase and cytochemical bioassays. ODS-silica extraction permitted examination of large plasms samples by reversed-phase h.p.l.c., facilitating the resolution of the various circulating molecular forms of parathyrin according to their hydrophobic character. Because of its rapidity, excellent recovery and high resolving power, the methodology utilized is uniquely suited to the purification and analysis of parathyrin in tissues and body fluids.

scholarly journals Determination of Nicotine in Libyan Smokers' Urine Compared with that of Nonsmokers using Reversed Phase – High Performance Liquid Chromatog-raphy (RP-HPLC)

2019 ◽  
Vol 34 (3) ◽  
pp. 172-180
Author(s):  
Galal Elmanfe ◽  
Suad K. Omar ◽  
Noreldin S.Y. Abdolla ◽  
Amna M. Hassan
Keyword(s):  
High Performance ◽  
Limit Of Detection ◽  
Cost Effective ◽  
High Performance Liquid Chromatog ◽  
Reversed Phase ◽  
Urine Samples ◽  
Average Value ◽  
Limit Of Quantitation ◽  
One Step

The aim of the present study was to evaluate the levels of nicotine in twenty urine samples taken from ten smokers and ten non-smokers in Libya. Each volunteer was required to complete a questionnaire before providing the urine sample. The evaluation of the nicotine concentrations was carried out by means of a simple, rapid, cost effective but reliable, one-step extraction technique-reversed-phase high-performance liquid chromatography which was developed and validated for this purpose. The criteria and factors taken into consideration for this evaluation and validation include the linearity, precision, accuracy, limit of detection, and limit of quantitation. The urine samples from the smokers presented nicotine concentrations in the range of 0.037-1.979 µg/ml, with an average of 0.663 µg/ml. The range of the nicotine concentrations in non-smokers, on the other hand, was from 0.017-1.331 g/ml, where 0.273 µg/ml is the average value. Statistical analyses show that the nicotine concentrations were very significant in the smoker samples in contrast with the nonsmoker samples

scholarly journals Development and Validation of a Novel Stability-Indicating Reversed-Phase High-Performance Liquid Chromatography Method for Assay of Loratadine and Determination of Its Related Compounds

2010 ◽  
Vol 93 (3) ◽  
pp. 891-903 ◽  
Author(s):  
Jun Lu ◽  
Yu-Chien Wei ◽  
Robert J Markovich ◽  
Abu M Rustum
Keyword(s):  
High Performance ◽  
Sodium Dodecyl ◽  
Active Pharmaceutical Ingredient ◽  
Reversed Phase ◽  
Hplc Method ◽  
Chromatography Method ◽  
Stability Indicating ◽  
The Stability ◽  
Related Compounds

Abstract Loratadine is an important active pharmaceutical ingredient used in a wide variety of prescription and over-the-counter products for the treatment and relief of allergy symptoms. A novel stability-indicating gradient ion-pair RP-HPLC method for assay of loratadine and determination of both of its degradation compounds and process impurities has been developed. This method can separate loratadine from its eight structurally related compounds; it can also separate all of the related compounds from each other in less than 20 min. The stability-indicating capability of this method has been demonstrated by analyzing aged stability samples of loratadine. A 15 cm 4.6 mm id YMC-Pack Pro C18 HPLC column was the primary column and a 15 cm 4.6 mm id SunFire C18 column has been identified as an alternate (truly equivalent) column for this method. This gradient method uses mobile phases consisting of acetonitrile and an aqueous solution of 10 mM sodium acetate and 5 mM sodium dodecyl sulfate at pH 5.5. The new HPLC method was validated according to International Conference on Harmonization guidelines and proved to be suitable for routine QC use.

A glyco-phosphoprotein in human milk

1987 ◽  
Vol 54 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Norihiro Azuma ◽  
Kunio Yamauchi
Keyword(s):  
Molecular Weight ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Gel Electrophoresis ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Reversed Phase ◽  
Ultracentrifugal Analysis

SummaryA highly glycosylated phosphoprotein (HGPP) was isolated from a human casein fraction by reversed-phase high-performance liquid chromatography. This component contained carbohydrates to ∼ 38·2% (w/w) and phosphorus to ∼ 1·6% (w/w). The molecular weight of this HGPP as estimated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis ∼ 41000. Ultracentrifugal analysis revealed that the sedimentation coefficient of the HGPP was 2·6S in a 10 mM-imidazole-HCl buffer at pH 7·0 and 27 °C, but this component interacted with human κ-casein and formed a complex with s = 10·4S.

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.

scholarly journals Eco-Innovation in Reusing Food By-Products: Separation of Ovalbumin from Salted Egg White Using Aqueous Two-Phase System of PEG 1000/(NH4)2SO4

Polymers ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 238 ◽  
Author(s):  
Bin Jiang ◽  
Jiaxin Na ◽  
Lele Wang ◽  
Dongmei Li ◽  
Chunhong Liu ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Egg White ◽  
Reversed Phase ◽  
Phase System ◽  
Ionization Mass ◽  
Two Phase ◽  
Aqueous Two Phase System

For the purpose of reducing pollution and the rational use of salted egg white, which is a byproduct of the manufacturing process of salted egg yolk, an aqueous two-phase system (ATPS) composed of polyethylene glycols (PEG 1000) and (NH4)2SO4 was investigated to selectively separate ovalbumin (OVA) from salted egg white. With the aim of optimizing the selective separation of OVA using ATPS, a response surface method (RSM) experiment was carried out on the basis of a single-factor experiment. The OVA was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), reversed-phase high-performance liquid chromatography (RP-HPLC), liquid chromatography-nano electrospray ionization mass spectrometry (Nano LC-ESI-MS/MS), and Fourier transform infrared spectroscopy (FT-IR). Under the optimal conditions, the recovery yield of OVA through ATPS (Y) and the purity of OVA (P) could reach 89.25% and 96.28%, respectively. In conclusion, OVA was successfully separated from the salted egg white by PEG/(NH4)2SO4 ATPS.

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