Gel filtration chromatography

Gel-filtration chromatography is a popular and versatile technique that permits the effective separation of proteins and other biological molecules in high yield. Here, the basis of the method is described and typical matrix types are contrasted. The selection of suitable operating conditions and applications of the method are also discussed

Obtaining Of β-Lactoglobulin By Gel Filtration Of Cow Milk Whey

Milk whey proteins carry out a number of important biological functions and also they are precursors of many biologically active peptides (antihypertensive peptides, antagonists of opioid receptors, regulators of intestinal motility, immunomodulatory, anti-microbial and anti-cancer peptides, appetite regulators and so on.). An important stage in natural bioactive peptides obtaining from milk whey proteins is the isolation of homogeneous proteins-precursors. Considering the significant difference in the molecular masses of whey proteins, a promising method for their selection is gel filtration. The purpose of the research was the fractionation of bioactive peptides precursors from milk whey using gel filtration on Sephadex G-150. The whey was obtained from fresh skimmed milk after isoelectric precipitation of casein. Gel filtration was carried out on the columns from a liquid chromatography kit by the “Reanal” company. The fractional composition and the degree of homogeneity of milk whey proteins were determined by disc-electrophoresis in the plates of a polyacrylamide gel. A repeated gel filtration of fractions from the chromatographic peaks, separated into sections, was performed to increase the fractionation efficiency. While choosing a dextran gel for gel filtration of precursors of biologically active peptides from milk whey proteins, we have taken into account the range of their molecular weights (from 10000 to 150000 Da), the ability to form supramolecular structures (β-LG), as well as the previously obtained results of gel filtration. As a result, it was shown that repeated gel filtration of milk whey on Sephadex G-150 allows efficiently fractionate the proteins-precursors of bioactive peptides. The range of peptides and proteins molecular weights that can be fractionated on this Sephadex is from 5000 to 300 000 Da. The usage of repeated gel filtration on Sephadex G-150 with the chromatogram separation into sectors allows to effectively fractionate proteins-precursors of bioactive peptides from milk whey. In particular, homogeneous β-lactoglobulin (degree of homogeneity > 95 %) and partially purified α-lactalbumin, as well as a group of immunoglobulins and a proteose-peptone fraction were obtained

A Monomer-to-Trimer Transition of the Human Mitochondrial Transcription Termination Factor (mTERF) Is Associated with a Loss of in Vitro Activity

The human mitochondrial transcription termination factor (mTERF) is a nuclear-encoded 39-kDa protein that recognizes a mtDNA segment within the mitochondrial tRNALeu(UUR) gene immediately adjacent to and downstream of the 16 S rRNA gene. Binding of mTERF to this site promotes termination of rDNA transcription. Despite the fact that mTERF binds DNA as a monomer, the presence in its sequence of three leucine-zipper motifs suggested the possibility of mTERF establishing intermolecular interactions with proteins of the same or different type. When a mitochondrial lysate from HeLa cells was submitted to gel filtration chromatography, mTERF was eluted in two peaks, as detected by immunoblotting. The first peak, which varied in proportion between 30 and 50%, appeared at the position expected from the molecular mass of the monomer (41 ± 2 kDa), and the gel filtration fractions that contained it exhibited DNA binding activity. Most interestingly, the material in this peak had a strong stimulating activity on in vitro transcription of the mitochondrial rDNA. The second peak eluted at a position corresponding to an estimated molecular mass of 111 ± 5 kDa. No mTERF DNA binding activity could be detected in the corresponding gel filtration fractions. Therefore, we propose that mTERF exists in mitochondria in two forms, an active monomer and an inactive large size complex. The estimated molecular weight of this complex and the fact that purified mTERF can be eluted from a gel filtration column as a complex of the same molecular weight strongly suggest that this inactive complex is a homotrimer of mTERF

Rubisco : easy purification and immunochemical determination

Rubisco (Ribulose-1.5-bisphosphate carboxylase/oxygenase) from spinach was purified to homogeneity in one step by gel filtration. This enzyme is suitable for the generation of a specific antibody in rabbits. The enzyme concentration in spinach leaves amounted to 40 % of the total soluble protein. The specific antibody shows cross reaction with crude extracts from leaves of other higher plants. The enzymes subunits could be separated by denaturing preparative SDS gel electrophoresis

Partial Purification of Histaminase from Guinea-Pig Liver by Gel-Filtration at High Temperature

Histaminase was partially purified by Sephadex G-200 gel filtration at high temperature. Guinea pig liver histaminase was extracted with heparin. The extract was fractionated by Sephadex G-200 gel filtration at 4°C. In the fractions containing histaminase, 7 dense and 2 faint protein bands were detected on SDS-gel electrophoresis. Further, fractionation of this sample by Sephadex G-200 gel filtration at 55°C markedly decreased the number of bands, i.e. only 1 dense and 2 faint bands were observed in the fractions in which histaminase activity was detected, and the enzyme activity was increased by approximately ten times. The results suggest that gel filtration at high temperature may be useful for partial purification of histaminase.This work was supported by Scientific Research Grant from the Japanese Ministry of Education (B, No. 58480264)

Copurification of actin and desmin from chicken smooth muscle and their copolymerization in vitro to intermediate filaments

Desmin is a 50,000-mol wt protein that is enriched along with 100-A filaments in chicken gizzard that has been extracted with 1 M KI. Although 1 M KI removes most of the actin from gizzard, a small fraction of this protein remains persistently insoluble, along with desmin. The solubility properties of this actin are the same as for desmin: they are both insoluble in high salt concentrations, but are solubilized at low pH or by agents that dissociate hydrophobic bonds. Desmin may be purified by repeated cycles of solubilization by 1 M acetic acid and subsequent precipitation by neutralization to pH 4. During this process, a constant nonstoichiometric ratio of actin to desmin is attained. Gel filtration on Ultrogel AcA34 in the presence of 0.5% Sarkosyl NL-97 reveals nonmonomeric fractions of actin and desmin that comigrate through the column. Gel filtration on Bio-Gel P300 in the presence of 1 M acetic acid reveals that the majority of desmin is monomeric under these conditions. A small fraction of desmin and all of the actin elute with the excluded volume. When the acetic acid is removed from actin-desmin solutions by dialysis, a gel forms that is composed of filaments with diameters of 120-140 A. These filaments react uniformly with both anti-actin and anti-desmin antiserum. These results suggest that desmin is the major subunit of the muscle 100-A filaments and that it may form nonstoichiometric complexes with actin

Purification of Mg2+-dependent phosphatidate phosphohydrolase from rat liver: new steps and aspects

A new procedure for the partial purification of Mg2+-dependent, N-ethylmaleimide-sensitive phosphatidate phosphohydrolase (Mg2+-PAP; EC 3.1.3.4) from rat liver cytosol is described, using protein precipitation with MgCl2, gel filtration on Sephacryl S-400, chromatography on DEAE-cellulose and affinity chromatography on calmodulin-agarose. From the parallel change in staining intensity and in the level of the specific activity of enzyme fractions, a relationship between a 90-kDa SDS gel band, identified as the beta-isoform of the 90-kDa heat shock protein, and Mg2+-PAP could be detected

Antioxidant activity in proteolytic enzyme digests of cashew (Anacardium occidentale L.) by-products

Membrane filtration of proteolytic enzyme digests and NaOH extracts of solvent extracted flours of cashew by-products resulted in reduction in antioxidant activities. Gel filtration of proteolytic enzyme digests of solvent extracted flours of cashew processing by-products on Sephadex G 25 revealed the presence of two peaks one immediately after the void volume and another later during elution. Reducing power, arginine and proteins content reduced in enzyme digests and alkali extracts after gel filtration and membrane filtration

Studies on the cornin extracted from bovine liver. I. Purification of the cornin and its physico-chemical properties

A factor, cornin, inhibiting the growth of L cells cultured in monolayer was extracted from bovine liver with boiling water and was partially purified by gel filtration with Sephadex G-200. The factor was (1) precipitable with ethanol at the concentration between 70% and 90%, (2) impermeable through dializing memo brane, (3) eluted as the last peak at the gel filtration and (4) containing protein and RNA but no DNA.</p

Numerical simulation of colloidal dispersion filtration: description of critical flux and comparison with experimental results

During filtration via membrane processes, colloids accumulate at the porous surface leading to fouling phenomena. In this study, a rigorous simulation of momentum and mass transfer using CFD modelling has been developed to describe such an accumulation during cross flow filtration. These simulations integrate detailed modeling of physicochemical properties specific to colloidal dispersions (because of the surface interactions (repulsive and attractive) occurring between the colloids particles). These interactions are accounted for via the experimental variation of the colloidal osmotic pressure with volume fraction (associated with a variation in the diffusion coefficient) which are fitted by a relationship integrated into the CFD code. It contains a description of the colloidal phase transition leading to the formation of a condensed phase (deposit or gel layer) from the accumulated dispersed phase (concentration polarization). It is then possible to determine the critical flux which separates filtration conditions below which mass accumulation is reversible (in the dispersed phase) and above which it is irreversible (in the condensed phase). The computed value of critical flux is compared with that determined experimentally for a dispersion of latex particles