Use of magnetic beads with immobilized monoclonal antibodies for isolation of highly pure plasma membranes

In plasma membrane proteome research, contamination of the isolated plasma membrane fraction with proteins from other organelles is still a problem. Even if highly specific isolation methods are used, such as density gradient centrifugation combined with selective extraction, contaminating proteins cannot be completely removed. To solve this problem, a protocol for the isolation of highly pure plasma membrane fractions from rat liver and two different hepatocellular carcinoma cell lines was developed. Magnetic beads with immobilized mAb's against highly expressed membrane proteins were used for specific binding of membrane vesicles and their separation from other organelles. Isolated plasma membranes were further selectively solubilized with different reagents and analyzed by use of different methods, such as Western blotting, 1- and 2-DE, and MS. Purification and further selective solubilization was validated by use of mAb's against the marker integral plasma membrane protein carcinoembryonic antigen cell adhesion molecule 1, and identification of isolated proteins by MS. The method presented here minimizes contamination with other organelles and enables further identification of membrane proteins.     

Review on recent advances in the analysis of isolated organelles

The analysis of isolated organelles is one of the pillars of modern bioanalytical chemistry. This review describes recent developments on the isolation and characterization of isolated organelles both from living organisms and cell cultures. Salient reports on methods to release organelles focused on reproducibility and yield, membrane isolation, and integrated devices for organelle release. New developments on organelle fractionation after their isolation were on the topics of centrifugation, immunocapture, free flow electrophoresis, flow field-flow fractionation, fluorescence activated organelle sorting, laser capture microdissection, and dielectrophoresis. New concepts on characterization of isolated organelles included atomic force microscopy, optical tweezers combined with Raman spectroscopy, organelle sensors, flow cytometry, capillary electrophoresis, and microfluidic devices.     

One-step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles

In the present study, we describe a new procedure using freezing-thawing to density gradient solution of Nycodenz for one-step separation of organelles from the rat liver and subsequent proteome analysis of subcellular fractions. To prepare two-dimensional electrophoresis (2-D PAGE) profiles of tissue organelles, we performed one-step subcellular fractionation of rat liver homogenate using a density gradient of Nycodenz solution, which resulted in the separation of the cytosolic fraction from the postnuclear supernatant. The density gradient of Nycodenz was prepared from a 20% solution in a centrifuge tube by freezing-thawing overnight at -20 degrees C and at room temperature for a few hours without the initial centrifugation procedure. The shape of the gradient density curve was dependent on Nycodenz concentration and tube size. After fractionation, the protein profiles were examined using one-dimensional sodium dodecyl sulfate (SDS)-PAGE. The organelles were confirmed using Western blotting. Our results indicate that our procedure provides a simple method for the separation of organelle fractions from the rat liver tissue.     

Interference study should be performed for every protein measurement method used

It is particularly important, when analyzing biological material, for the measurement procedure to be specific to the analyte and not to suffer interference by the matrix effect. Tissue fraction studies also require rapid and accurate methods to estimate the concentration of protein in solutions as well as many measurement methods used in medical laboratories. The design of this study is based on a comparison of the Lowry and the bicinchoninic acid (BCA) methods for the measurement of the total protein concentrations of rat liver subcellular fractions. In our experiment, subcellular fractions enriched in peroxisomes (POs) obtained by differential centrifugation were then further separated by means of density gradient centrifugation. We performed the protein measurement assays on all fractions obtained during the purification steps. The protein contents of the fractions obtained were determined by the two methods. The method comparison statistics were performed by linear Deming regression analysis and Altman and Bland bias plot. The regression equation was unacceptable, indicating that the last three fractions separated by means of Nycodenz discontinuous density gradient centrifugation gave remarkably divergent results. For the Lowry method, the Nycodenz effect could not be eliminated with the use of interference blank. In addition to Nycodenz, the potentially interfering compound used in the isolation procedure as isolation medium was 3-(morpholino)propane sulfonic acid (MOPS). In decreased concentrations of MOPS (10 mM), interference blank should be used for correct measurement with Lowry, but in practical use 10 mM does not provide buffering potency. In the BCA method, interference blank correction seemed to eliminate the measurement error in all concentrations of Nycodenz. There was no MOPS effect on the BCA measurement assay. Referring to deviations as sample-inherent matrix effects, we concluded that not only one, but more measurement methods should be used in order to make a correct protein measurement.     

Effects of peptide density and elution pH on affinity chromatographic purification of human immunoglobulins A and M

A family of linear hexamer peptide ligands HWRGWV, HYFKFD and HFRRHL, initially identified for their affinity to the Fc portion of human immunoglobulin G (hIgG), also have potential for use in the purification of human immunoglobulins A (hIgA) and M (hIgM). HWRGWV demonstrated the strongest binding affinity to hIgM, followed by hIgA and hIgG respectively. The effects of N-terminal acetylation of the peptide, as well as elution buffer pH, on the chromatographic elution of human IgG, IgA and IgM from HWRGWV resins at various peptide densities (0.04-0.55 meq/g) were investigated. Over 80% recovery and 90% purity were achieved for human IgG and IgA isolation from complete minimum essential medium (cMEM) using HWRGWV resin at optimum peptide densities. For human IgM, 75.7% recovery and 86.0% purity were achieved by using HWRGWV at a low peptide density of 0.04 meq/g. Although HYFKFD and HFRRHL exhibited their ability for isolation of human IgG, IgA and IgM from cMEM as well, HWRGWV is the best option among them for large-scale purification of human IgG, IgA and IgM based on conditions tested.     

The isolation and purification of diamine oxidase of pea seedlings and pig liver

Enzymes have been extensively used in many industries for the last 20 years. They are becoming more common because of new areas of application. The limitations of applications of enzymes are activity, specificity, stability, and price. Higher enzyme activities and less susceptibility of process conditions are desirable. Therefore, in some cases, purified enzyme extracts are needed. The purpose of this study is the isolation and purification of diamine oxidase (DAO) of pea seedlings and pig liver. The relationship between pea seedlings growth rate and enzyme activity is established. DAO of pea seedlings and pig liver is prepared by way of tissue disruption with homogenization, centrifugation, fractionation with ammonium sulfate, precipitation of inert components, column electrophoresis, and DEAE-cellulose column chromatography. The specific activity of disrupted pea seedlings cells was measured as 0.017 (U/mg protein), and the pig liver DAO activity was measured as 0.00037 (U/mg protein). The specific enzyme activity from pea seedlings was increased to 6.750 (U/mg protein). On the other hand, the specific enzyme activity from pig liver was increased to 0.30 (U/mg protein). The final enzyme extract from pea is 400-fold purer than raw material, and the final enzyme extract from pig liver is 820-fold purer than raw material.     

Selective elution of soluble rat liver glutathione transferases from a glutathione-Sepharose affinity column

Glutathione transferases (GST) are dimeric enzymes that take part in many detoxification processes. A previous report described the use of a glutathione-Sepharose affinity matrix for the purification of human liver GST. The method involved the use of 5 mM glutathione in a high pH buffer, and the yields were nearly 100%. This method and adapted techniques have now been applied to rat liver GST. Selective GST elution can be obtained in several different ways: by stepwise change of the pH and/or glutathione concentration, and by linear gradient elution. Gel electrophoresis showed, however, that none of the fractions contained pure GST isoenzymes. Also, less than 50% of the total rat liver GST was eluted with 5 mM glutathione, in contrast to the results with human liver GST. A glutathione concentration of 30 mM is necessary for quantitative desorption of rat liver GST from a glutathione-Sepharose column.     

Analysis and purification of DNA restriction fragments by high-performance liquid chromatography.

The purification and analysis of restriction fragments play a very important role in molecular biology but the traditional assay methods of DNA fragments, based on gel electrophoresis and caesium chloride gradient centrifugation, are time-consuming and difficult to quantify. High-performance liquid chromatography provides an alternative method which allows the direct quantitation of picogram amounts of eluents in short time. In the present work we report the separation of different restriction fragments, the purification of some fragments and the relationship between the length of double-stranded DNA fragments and peak areas.     

Immobilized pH gradient isoelectric focusing and immunoblotting for investigations of anti-Borrelia burgdorferi IgG antibodies.

Anti-Borrelia burgdorferi immunoglobulin G (IgG) responses in cerebrospinal fluid, serum, and joint fluid from Lyme disease patients were investigated by immobilized pH gradient (IPG) isoelectric focusing (IEF) in pH 4-10 and pH 4-7 gels. After focusing, the anti-B.-burgdorferi antibodies were blotted by affinity-driven transfer to antigen-coated polyvinylidene difluoride membranes (immunoblot) and the IgG antibodies were immunoenzymatically stained. IPG-IEF gels gave an excellent resolution of IgG and the immunoblot proved advantageous for the detection of anti-B. burgdorferi IgG antibodies. These antibodies, as judged from the electromigration characteristics, were found to contain oligoclonal as well as polyclonal subpopulations. This latter group included IgG antibodies that were inadequately resolved when separated by conventional carrier ampholyte IEF.     

猪肝中单胺氧化酶B的分离纯化

依据单胺氧化酶B(monoamine oxidase B, MAOB)的疏水特性,建立了一种从猪肝中分离纯化MAOB的新方法。用含有1% Triton X-100的膜蛋白裂解液制备粗酶,以饱和度为20%～50%的硫酸铵反抽提进行粗提,再利用自制的配基密度为75.7 μmol/mL的苯基疏水色谱及Sepharose Q High Performance离子交换色谱进一步分离纯化,得到纯化倍数为18.2、酶比活为135 U/mg的MAOB。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示为相对分子质量约60 000的单一蛋白质带。采用高效液相色谱-电喷雾串联质谱对该酶进行鉴定,证实为MAOB。本研究所用分离纯化方法可以有效纯化MAOB, 为MAOB的深入研究提供技术支撑。     

Scale-up of free flow electrophoresis: I. Purification of alcohol dehydrogenase from a crude yeast extract by zone electrophoresis

The potential and limitations in scaling-up free-flow electrophoresis, with emphasis on zone electrophoresis, are demonstrated. Purification of alcohol dehydrogenase (ADH) from a crude yeast extract was chosen as a model for an industrial approach to enzyme purification. In zone electrophoresis the separation quality strongly depends on the pH and conductivity of the background electrolyte, its residence time and flow rate, as well as the applied voltage. Optimization of these parameters resulted in a purification factor of 5.3 and a yield of 96% ADH, using a Tris/HCl buffer, pH 8.0, and a conductivity of 1 mS/cm, with a residence time of 10 min at 500 V. The loading capacity of the method for a laboratory-sized free-flow electrophoresis apparatus was limited to a sample throughput of about 0.4 g/h. By increasing the chamber dimensions it was possible to purify the enzyme by a purification factor of 4.7 and a yield of 93% ADH, at a throughput of about 1 g total protein/h. By simultaneously applying the sample at 3 input positions the throughput could be increased to 2.75 g/h with a purification factor of 4.7 and an overall yield of 90%.     

Optimization of the mobile phase conditions for the purification ofTaql endonuclease

Summary Taql endonuclease was purified by high performance ion exchange liquid chromatography with linear gradient elution. Of the chromatographic media tested as mobile phases for the HPLC purification ofTaql endonuclease, sodium acetate (pH 5.0) and phosphate (pH 7.0) buffers were appropriate for use with cation exchange columns, and L-histidine (pH 6.0) and Tris (pH 8.0) buffers with anion exchange columns.     

Surfactant-aided size-exclusion chromatography for the purification of immunoglobulin G

In the production of monoclonal antibodies, separate chains of the antibody are often present in the product mixture as well as other contaminating proteins. These fragments should be removed from the whole antibodies. This paper shows the purification of monoclonal immunoglobulin G (IgG) from its heavy chain contaminant. The heavy chain fragment is simulated experimentally using bovine serum albumin (BSA), which has approximately the same molecular weight. The purification is performed using traditional size-exclusion chromatography (SEC) and using surfactant-aided SEC (SASEC), testing two different surfactants (C(12)E(23) and Tween20) and two different gels (Sephacryl S200HR and Sephacryl S300 HR). Pulse experiments show that with SASEC both BSA and IgG are more distributed towards the solid phase than compared to using SEC. This effect is larger on IgG, the largest component than on BSA. As a consequence, azeotropes will be formed at a specific surfactant concentration. Above this concentration the selectivity is reversed and increased to values higher than obtained with conventional SEC. At 7.5% (w/w) of C(12)E(23), BSA actually elutes before IgG. These experiments further show that when using SASEC larger productivity, higher yields and lower solvent consumption can be achieved without loss of purity of IgG when compared to conventional SEC. Mathematical simulation of the separation of BSA and IgG using simulated moving bed (SMB) chromatography indicates a large increase in productivity when applying a surfactant gradient in SASEC SMB compared to conventional isocratic SEC-SMB. Furthermore, solvent consumption reductions with a factor 15 prove possible as well as concentrating the IgG by a factor 2.     

Purification and Properties of the diamine oxidase of pea seedlings

Enzymes have been used extensively in many industries for the last 20 yrs. The purpose of this study was the isolation, purification, and specification of diamine oxidase (DAO) of pea seedlings. The relationship between enzyme activity and growth conditions has been investigated. DAO that was extracted from pea seedlings was purified by centrifugation, thermal denaturation, fractionation with ammonium sulfate, precipitation of inert components, column electrophoresis, and DEAE-cellulose column chromatography. It was found that the final enzyme preparation is 400-fold purer than the original extract at the end of the purification steps. The molecular weight, isoelectric point, and copper content of the purified enzyme also were determined.     

The rat liver mitochondrial proteins

Subcellular fractionation increases the probability of detection of low-abundance proteins. We prepared a fraction highly enriched in mitochondrial proteins from rat liver. The proteins were analyzed by two-dimensional (2-D) electrophoresis using broad-and narrow-range immobilized pH gradient strips, and identified by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). 192 different gene products were detected, of which approximately 70% were enzymes with a broad spectrum of catalytic activities. Most of the identified proteins were detected in other rat protein samples as well, which were analyzed in our laboratory. Eight gene products were detected for the first time. These were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. In average, approximately 10-15 spots corresponded to one gene product.     

A rapid method for fractionation and purification of a 92 kDa cartilage glycoprotein

Matrix glycoproteins are among the main components that contribute to the properties of cartilage. In this article we report on the development of a rapid method for the fractionation and purification of a 92 kDa glycoprotein from chick sternal cartilage. The developed procedure involves ion-exchange chromatography on DEAE-Sephacel, gel permeation chromatography on Sepharose CL-6B and semi-preparative SDS-polyacrylamide gel electrophoresis. Identification of protein was performed by western blotting using specific antibodies and purity by capillary electrophoresis. The proposed method is superior to those previously published since it eliminates the step of density gradient centrifugation.     

Fast electrophoretic analysis of individual mitochondria using microchip capillary electrophoresis with laser induced fluorescence detection

The analysis of mitochondria by capillary electrophoresis usually takes longer than 20 min per replicate which may compromise the quality of the mitochondria due to degradation. In addition, low sample consumption may be beneficial in the analysis of rare or difficult samples. In this report, we demonstrate the ability to analyze individual mitochondrial events in picoliter-volume samples (approximately 80 pL) taken from a bovine liver preparation using microchip capillary electrophoresis with laser-induced fluorescence detection (micro-chip CE-LIF). Using a commercial "double-T" glass microchip, the sample was electrokinetically loaded in the "double-T" intersection and then subjected to electrophoretic separation along the main separation channel. In order to decrease interactions of mitochondria with channel walls during the analysis, poly(vinyl alcohol) was used as a dynamic coating. This procedure eliminates the need for complicated covalent surface modifications within the channels that were previously used in capillary electrophoresis methods. For analysis, mitochondria, isolated from bovine liver tissue, were selectively labelled using 10-nonyl acridine orange (NAO). The results consist of electropherograms where each mitochondrial event is a narrow spike (240 +/- 44 ms). While the spike intensity is representative of its NAO content, its migration time is used to calculate and describe its electrophoretic mobility, which is a property still largely unexplored for intracellular organelles. The five-fold decrease in separation time (4 min for microchip versus 20 min for capillary electrophoresis) makes microchip electrophoretic separations of organelles a faster, sensitive, low-sample volume alternative for the characterization of individual organelle properties and for investigations of subcellular heterogeneity.     

One-step separation of endocytic organelles, Golgi/trans-Golgi network and plasma membrane by density gradient electrophoresis

Many different methods for the fractionation of subcellular organelles have been reported. However, no protocol for rapid separation of plasma membrane, Golgi/trans-Golgi network (TGN) and endosomes is available to date. Such a method is a prerequisite for a quantitative biochemical analysis of vesicular transport from the Golgi/TGN compartment to plasma membrane and endosomes. Here a density gradient electrophoresis protocol is described that allows the fractionation of these organelles in one step. This protocol requires only low-cost instrumentation available in most biochemical laboratories.     

4-(1H-imidazol-1-yl) aniline: A new ligand of mixed-mode chromatography for antibody purification

4-(1H-imidazol-1-yl) aniline (AN) was immobilized on Sepharose CL-6B (AN-Sepharose) for use as a new ligand of mixed-mode chromatography. Adsorption equilibria of immunoglobulin G (IgG) and bovine serum albumin (BSA) to AN-Sepharose were studied at extensive pH values (4.0–8.8) and salt concentrations (0–1.0 mol/L). Static binding studies indicated that AN-Sepharose had a good salt-tolerance property for IgG adsorption up to 1.0 mol/L NaCl. This was attributed to the combined ligand–protein interactions (hydrophobic interaction, hydrogen bonding and charge transfer interaction). By contrast with BSA, AN-Sepharose showed a high binding selectivity for IgG at NaCl > 0.2 mol/L. Dynamic binding capacities (DBC) of IgG and BSA at 10% breakthrough were measured at pH 4.0–8.8 by frontal analysis chromatography. IgG had DBC values over 40 mg/mL at pH 7.0–8.8, and the maximum reached 59 mg/mL at pH 8.0. At pH 5.0, a distinct drop in DBC to 8.5 mg/mL was observed, but that for BSA kept over 22 mg/mL. The result suggested that IgG could be selectively desorbed from AN-Sepharose by decreasing pH to about 5. Therefore, compared to BSA, AN-Sepharose exhibited a dual-selectivity for IgG in both adsorption and elution. Purification of IgG from bovine serum also confirmed the dual-selectivity. IgG purity of the pooled fractions by elution at pH 4.0, 4.5 and 5.0 reached 55% and the highest purity, 80%, was obtained at pH 4.5. The average purification factor of IgG was over 25. The results indicate that AN is a promising ligand of mixed-mode chromatography for antibody purification from a complex feedstock.