[22] Precipitation techniques

Englard, Sasha; Seifter, Sam

doi:10.1016/0076-6879(90)82024-v

Cited by 281 publications

(153 citation statements)

References 6 publications

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“…As the concentration of organic solvent is increased, attraction between macromolecules increases, the solvent becomes less effective (the activity coefficient of water is reduced) and the solid state is favored (Cohn et al, 1974;Englard & Seifter, 1990). Organic solvents should be used at a low temperature, at or below 0 C, and they should be added very slowly with good mixing (McPherson, 1999).…”

Section: Figurementioning

confidence: 99%

Introduction to protein crystallization

McPherson

2004

Methods

240

201

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Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies.

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Section: Figurementioning

confidence: 99%

Introduction to protein crystallization

McPherson

2004

Methods

240

201

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“…Aqueous extracts were precipitated with ammonium sulfate ([NH 4 ] 2 SO 4 ) according to Englard and Seifter [25]. The precipitaton was carried out eliminating first the protein fraction precipitated at 40% of ammonium sulfate saturation, and then collecting the fraction that precipitated at 60% ammonium sulfate saturation.…”

Section: Lectin Purification and Agglutinating Activity Determinationmentioning

confidence: 99%

Characterization of Two Non-Fetuin-Binding Lectins from Tepary Bean (Phaseolus acutifolius) Seeds with Differential Cytotoxicity on Colon Cancer Cells

Arteaga¹,

Guillen²

2016

J Glycobiol

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Two non-fetuin-binding lectins from tepary bean (Phaseolus acutifolius) seeds were purified and characterized. Both lectins LA and LB showed by two dimensional gel electrophoresis very similar apparent molecular mass, and their isoelectric point for both lectins were also very close. Mass spectrometry analysis confirmed these similarities showing high sequence homology among them, and when these sequences were compared to the Mirkov´s deduced sequence of the phyto-hemagglutinin from Phaseolus acutifolius, LA showed higher homology than LB. On the other hand, when their biological activity was determined, only LA possessed affinity to type A erythrocytes, and when these lectins using a semi pure fraction were measured for their cytotoxic effect on colon cancer cells, they showed a differential effect. In the semi pure protein fraction tested, the rest of no-lectin proteins separated by electrophoresis presented no activity. Our results indicate that despite the high homology in their sequence, isoelectric point, and apparent molecular mass, these two structurally closely related lectins, showed differences in their biological behaviour, represented by their agglutinatination activity, as well as, in their cytotoxic effect on human colon cancer cells.

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“…Generally, methods for protein purification include salt precipitation, [1,2] solvent precipitation, [2] ion-exchange, and affinity separation. Among these methods, affinity separation is superior to the other methods because of the process efficiency and because the biomolecules are recovered in high purity.…”

Section: Introductionmentioning

confidence: 99%

Thermoprecipitation of Glutathione S‐Transferase by Glutathione–Poly(N‐isopropylacrylamide) Prepared by RAFT Polymerization

Chang

Nguyen

Maynard

2010

Macromol. Rapid Commun.

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Herein, we report an effective and rapid method to purify glutathione S‐transferase (GST) using glutathione (GSH)‐modified poly(N‐isopropylacrylamide) (pNIPAAm) and mild, thermal conditions. A chain transfer agent modified with pyridyl disulfide was employed in the reversible addition–fragmentation chain transfer (RAFT) polymerization of NIPAAm. The resulting polymer had a narrow molecular weight distribution (polydispersity index = 1.21). Conjugation of GSH to the pyridyl disulfide–pNIPAAm reached 95% within 30 min as determined by UV–Vis monitoring of the release of pyridine‐2‐thione. GST was successfully thermoprecipitated upon heating the GSH–pNIPAAm above the lower critical solution temperature (LCST). The pull down assay was repeated with bovine serum albumin (BSA) and T4 lysozyme (T4L), which demonstrated the specificity of the polymer for GST. Due to its simplicity and high efficiency, this method holds great potential for large‐scale purification of GST‐tagged proteins.magnified image

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[22] Precipitation techniques

Cited by 281 publications

References 6 publications

Introduction to protein crystallization

Introduction to protein crystallization

Characterization of Two Non-Fetuin-Binding Lectins from Tepary Bean (Phaseolus acutifolius) Seeds with Differential Cytotoxicity on Colon Cancer Cells

Thermoprecipitation of Glutathione S‐Transferase by Glutathione–Poly(N‐isopropylacrylamide) Prepared by RAFT Polymerization

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