Isolation and Purification of Jacalin from Artocarpus Heterophyllus Lam

This paper presents investigation results of saturation conditions needed for purification of jacalin lectin from the extract seeds of Artocarpus heterophyllus by ammonium precipitation and affinity chromatography on Galactose-Affi gel Hz. Three different aspects of parameters encompassing the percentage of saturation of ammonium sulfate precipitation, the presence of ammonium sulfate on Lowry method and the suitable galactose concentration for optimum elution of the protein from Galactose-Affi gel Hz were investigated. With three different sets of fractional saturation of jacalin purification using ammonium sulfate precipitation, the maximum yield of 0.463 g/g was achieved at 0-90% saturation range in the absence of dialysis. Maximum yield of 0.425 g/g was obtained at 30-60% and 0-90% saturation range in the presence of dialysis. The result from this work also indicates that excessive quantity of NH4SO4 interferes with Lowry method for protein determination substantially. The 0-90% saturation range was found to be more potentially appropriate for large scale application than 30-60% saturation, since the former involves only 1 step NH4SO4 addition. From the affinity chromatography, elution of 0.2 M galactose (in 0.15 M NaCl) from Galactose-Affi gel Hz produced the maximum peak profile and jacalin concentration. A reduction or increase in galactose concentration of more than 0.2 M did not increase concentration of purified jacalin purified using this method.

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A Simple, Large Scale Process for Purifying Human Urinary Kallikrein, Based on Reverse Osmosis and Ammonium Sulfate Precipitation

Kinins—II - Advances in Experimental Medicine and Biology ◽

10.1007/978-1-4757-0926-1_29 ◽

1979 ◽

pp. 305-312 ◽

Cited By ~ 2

Author(s):

Amintas F. S. Figueiredo ◽

Marcos Mares-Guia

Keyword(s):

Ammonium Sulfate ◽

Reverse Osmosis ◽

Large Scale ◽

Urinary Kallikrein ◽

Ammonium Sulfate Precipitation

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A GENERAL METHOD FOR THE ISOLATION AND PURIFICATION OF PHOSVITIN FROM VERTEBRATE EGGS

Canadian Journal of Biochemistry ◽

10.1139/o66-187 ◽

1966 ◽

Vol 44 (12) ◽

pp. 1647-1655 ◽

Cited By ~ 90

Author(s):

R. A. Wallace ◽

D. W. Jared ◽

A. Z. Eisen

Keyword(s):

Molecular Weight ◽

Ammonium Sulfate ◽

Deae Cellulose ◽

The Other ◽

Chemical Analyses ◽

Ammonium Sulfate Precipitation ◽

Isolation And Purification ◽

Complex Ii ◽

Vertebrate Eggs ◽

General Method

A general method has been developed for the isolation and purification of phosvitin from vertebrate eggs. The method is detailed in three steps consisting of (i) isolation of a phosvitin–lipovitellin complex; (ii) ammonium sulfate precipitation of the lipovitellin; and (iii) DEAE-cellulose chromatography of the remaining phosvitin. Phosvitin was isolated from the eggs of five representative vertebrates (lamprey, trout, frog, turtle, and chicken), and chemical analyses together with sedimentation studies were performed on the samples. Preparations were obtained with the lowest N/P ratios reported to date. The analytical results also suggested that trout phosvitin has approximately half the molecular weight (24,000) of the other proteins and that "purified" phosvitin may still be heterogeneous.

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Abundant plasma protein depletion using ammonium sulfate precipitation and Protein A affinity chromatography

Journal of Chromatography B ◽

10.1016/j.jchromb.2018.04.045 ◽

2018 ◽

Vol 1089 ◽

pp. 43-59 ◽

Cited By ~ 6

Author(s):

Lentel Pringels ◽

Valérie Broeckx ◽

Kurt Boonen ◽

Bart Landuyt ◽

Liliane Schoofs

Keyword(s):

Affinity Chromatography ◽

Ammonium Sulfate ◽

Plasma Protein ◽

Protein A ◽

Ammonium Sulfate Precipitation ◽

Protein Depletion

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THE ISOLATION AND PURIFICATION OF A TRYPSIN INHIBITOR FROM WHOLEWHEAT FLOUR

Canadian Journal of Biochemistry ◽

10.1139/o64-194 ◽

1964 ◽

Vol 42 (12) ◽

pp. 1825-1832 ◽

Cited By ~ 25

Author(s):

G. Shyamala ◽

R. L. Lyman

Keyword(s):

Ammonium Sulfate ◽

Trypsin Inhibitor ◽

Carboxymethyl Cellulose ◽

Moving Boundary ◽

Specific Activity ◽

Trypsin Inhibitors ◽

Ammonium Sulfate Precipitation ◽

Commercial Sample ◽

Isolation And Purification ◽

Single Protein

An impure concentrate of a trypsin inhibitor was prepared from a commercial sample of wholewheat flour by ammonium sulfate precipitation. The impure inhibitor was heat-labile and did not inhibit pepsin or α-chymotrypsin activity. When the crude inhibitor was chromatographed on carboxymethyl cellulose, a single protein peak that was about 20 times more active than the original impure inhibitor preparation was obtained.Uultracentrifugal sedimentation patterns and moving-boundary electrophoresis indicated that the material isolated on the carboxymethyl cellulose was nearly homogeneous. In comparison with other known trypsin inhibitors that have been isolated in nearly pure form, wholewheat trypsin inhibitor appears to have a low specific activity.

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Proteinases from buckwheat (Fagopyrum esculentum moench) seeds: Purification and properties of the 47 kDa enzyme

Archives of Biological Sciences ◽

10.2298/abs0603171t ◽

2006 ◽

Vol 58 (3) ◽

pp. 171-177 ◽

Cited By ~ 5

Author(s):

Gordana Timotijevic ◽

Svetlana Radovic ◽

Vesna Maksimovic

Keyword(s):

Affinity Chromatography ◽

Ammonium Sulfate ◽

Proteolytic Activity ◽

Membrane Fraction ◽

Aspartic Proteinase ◽

Ammonium Sulfate Precipitation ◽

Milk Clotting ◽

Purification And Properties ◽

Fagopyrum Esculentum Moench ◽

Pepstatin A

Aspartic proteinases from buckwheat seeds are analyzed. Three forms of 47 kDa, 40 kDa and 28 kDa, were purified from mature buckwheat seeds, while two forms of 47 kDa and 28 kDa were detected in developing buckwheat seeds using pepstatin A affinity chromatography. A form of 47 kDa was selectively precipitated from other forms by ammonium sulfate precipitation. This enzyme resembles the chymosin-like pattern of proteolytic activity, as it was shown using BSA and k-casein as substrates, clarifying its ability for milk-clotting. The 47 kDa aspartic proteinase form is localized in the membrane fraction. .

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Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128900 ◽

1987 ◽

Vol 45 ◽

pp. 924-925

Author(s):

F. A. Durum ◽

R. G. Goldman ◽

T. J. Bolling ◽

M. F. Miller

Keyword(s):

Inclusion Bodies ◽

Large Scale ◽

Recombinant Dna ◽

Test System ◽

Cell Protein ◽

Gram Negative Bacteria ◽

Cytoplasmic Inclusions ◽

Isolation And Purification ◽

E Coli ◽

Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

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Extraction and Partial Purification of Protease from Bilimbi (Averrhoa bilimbi L.)

Scientific Research Journal ◽

10.24191/srj.v10i2.9403 ◽

2013 ◽

Vol 10 (2) ◽

pp. 29

Author(s):

Normah Ismail ◽

Nur' Ain Mohamad Kharoe

Keyword(s):

Molecular Weight ◽

Protein Content ◽

Ammonium Sulfate ◽

Molecular Weight Distribution ◽

Weight Distribution ◽

Protease Activity ◽

Protein Concentration ◽

Temperature Stability ◽

Partial Purification ◽

Ammonium Sulfate Precipitation

Unripe and ripe bilimbi (Averrhoa bilimbi L.) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity of both the unripe and ripe fruit were optimum at pH 4 and 40°C when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between IO to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage.

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A set of simple methods for detection and extraction of laminarinase

Scientific Reports ◽

10.1038/s41598-021-81807-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ananthamurthy Koteshwara ◽

Nancy V. Philip ◽

Jesil Mathew Aranjani ◽

Raghu Chandrashekhar Hariharapura ◽

Subrahmanyam Volety Mallikarjuna

Keyword(s):

Ammonium Sulfate ◽

Ammonium Sulphate ◽

Gold Standard ◽

Direct Detection ◽

Low Cost ◽

Direct Analysis ◽

Reducing Sugar ◽

Ammonium Sulfate Precipitation ◽

Simple Method ◽

Functional Step

AbstractA carefully designed ammonium sulfate precipitation will simplify extraction of proteins and is considered to be a gold standard among various precipitation methods. Therefore, optimization of ammonium sulfate precipitation can be an important functional step in protein purification. The presence of high amounts of ammonium sulphate precludes direct detection of many enzymatically active proteins including reducing sugar assays (e.g. Nelson-Somogyi, Reissig and 3,5-dinitrosalicylic acid methods) for assessing carbohydrases (e.g. laminarinase (β (1–3)-glucanohydrolase), cellulases and chitinases). In this study, a simple method was developed using laminarin infused agarose plate for the direct analysis of the ammonium sulphate precipitates from Streptomyces rimosus AFM-1. The developed method is simple and convenient that can give accurate results even in presence of ammonium sulfate in the crude precipitates. Laminarin is a translucent substrate requiring the use of a stain to visualize the zones of hydrolysis in a plate assay. A very low-cost and locally available fluorescent optical fabric brightener Tinopal CBS-X has been used as a stain to detect the zones of hydrolysis. We also report simple methods to prepare colloidal chitin and cell free supernatant in this manuscript.

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