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1.
The chromatographic separation of an unstable protein is often a challenge to the scientist working in the field of life sciences. Especially for the purification of sensitive enzymes, making use of conventional chromatographic techniques is difficult and frequently results in a complete loss of biological activity of the target protein. This report summarizes some general strategies that may help to keep unstable proteins in their native conformation during the rather harsh conditions of a purification procedure. In this context, a recently developed hollow fiber membrane module, suitable for performing on-line dialysis, is introduced and examples of its application to liquid column chromatography are given. Many innovative separation techniques, characterized by dramatic improvements in both performance and separation time, have recently been developed. Since the chromatographic separation of unstable proteins requires the use of modern state-of-the-art equipment and technology, emphasis is given to newly developed separation techniques such as expanded bed adsorption, perfusion chromatography, protein free flow electrophoresis and the use of tentacle gels. In addition, examples of recently published purifications of unstable proteins are discussed with respect to strategies ensuring the preservation of the native protein structure during chromatographic separation.  相似文献   

2.
We assessed the BioFocus 2000 capillary electrophoresis instrument for use in a routine clinical laboratory. We examined 210 serum samples received for serum protein electrophoresis by four methods: (1) The Bio-Rad HR015EC high-resolution serum protein kit on the BioFocus; (2) the Jenkins–Guerin (JG) method on the Applied Biosystems 270A HT Capillary Electrophoresis System (JG-ABI); (3) the Jenkins–Guerin method using the BioFocus (JG-BF); and (4) the quantitation of monoclonal bands found in 76 of the 210 samples was assayed by Helena Titan Hi-Res agarose gel electrophoresis (HRAGE). The correlation coefficient between the three sets of capillary electrophoresis monoclonal band results and the Helena quantitation was 0.92 or better. Although the quantitative comparison of monoclonal bands by HR015EC was very good, the lack of sharpness of monoclonal bands using the HR015EC kit meant our preference was to use the JG method on either the ABI or on the Biofocus.  相似文献   

3.
Different analytical techniques involving capillary electrophoresis for the determination of drugs and metabolites in biological fluids are described. Pharmacokinetic studies carried out using capillary electrophoresis are presented, as well as the in vitro metabolism investigations. The advantages and the limitations of capillary electrophoresis for pharmacokinetic studies are discussed.  相似文献   

4.
Recent developments in capillary electrophoresis (CE) in conjunction with laser-induced fluorescence (LIF) using long-wavelength (maximum excitation wavelength>500 nm) dyes are reviewed. These dyes are particularly of interest when conducting the analyses of biopolymers by CE-LIF using He-Ne lasers. These systems are benefited from low background, low costs, easy maintenance, and compactness. Derivatizations of DNA and proteins with fluorescent or nonfluorescent chemicals can be carried out prior to, during, or after separations. With the advantages of sensitivity, rapidity, and high efficiency, the applications of CE-LIF to the analysis of polymerase chain reaction products, DNA sequencing, trace analysis of proteins, and single cell analysis have been presented.  相似文献   

5.
Microdialysis sampling has become an important method for the continuous monitoring from an in vivo environment. This technique has been used to monitor many endogenous molecules, such as neurotransmitters, as well as exogenous species such as drug substances. Microdialysis samples have traditionally been analyzed by liquid chromatographic (LC) methods to gain resolution and quantification of the molecules of interest. However, LC separations have a relatively large injection volume requirement which, as a consequence, increases microdialysis sampling times. Capillary electrophoresis (CE), with its very small sample volume requirements and high resolving power, has therefore gained popularity as an alternative to LC. Reviewed here are many of the technologies currently available for CE and examples of how this technique has been effectively applied to the analysis of microdialysis samples.  相似文献   

6.
High-performance affinity chromatography (HPAC) is a method in which a biologically-related ligand is used as a stationary phase in an HPLC system. This approach is a powerful means for selectively isolating or quantitating agents in complex samples, but it can also be employed to study the interactions of biological systems. In recent years there have been numerous reports in which HPAC has been used to examine the interactions of drugs, hormones and other substances with serum proteins. This review discusses how HPAC has been used in such work. Particular attention is given to the techniques of zonal elution and frontal analysis. Various applications are provided for these techniques, along with a list of factors that need to be considered in their optimization and use. New approaches based on band-broadening studies and rapid immunoextraction are also discussed.  相似文献   

7.
In theory, peptide mass fingerprinting by matrix assisted laser desorption–ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower μl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.  相似文献   

8.

Background  

Numerous gel-based softwares exist to detect protein changes potentially associated with disease. The data, however, are abundant with technical and structural complexities, making statistical analysis a difficult task. A particularly important topic is how the various softwares handle missing data. To date, no one has extensively studied the impact that interpolating missing data has on subsequent analysis of protein spots.  相似文献   

9.
10.
The binding protein to a hypoglycosylated IgA1/Sepharose (IgA1-BP) could be prepared from human sera. IgG was a major component in the IgA1-BP. A Protein A column was used to remove the IgG; however, about half of the IgA1-BP was passed from the column [Biochem. Biophys. Res. Commun., 264 (1999) 424]. Quantitative analysis of the passed fraction (PAP) by laser nepherometry indicated that it was composed of a fairly large amount of IgA, IgM and complement C3 besides IgG. The relative content of IgG:IgA:IgM:C3:C4 was 25:10:41:22:2 in the PAP fraction. Meanwhile, the Protein A bound-fraction was essentially composed of IgG (78%) and IgM (19%). The total amount of IgA1-BP was not different between the sera from IgA nephropathy patients and other nephropathy patients. With respect to the IgA content in the IgA1-BP from IgA nephropathy patients, it was significantly higher than that from other nephropathy patients. It was found that the IgA1-BP from some IgA nephropathy patients contained a few micrograms of aberrant IgA per ml of serum. Thus, the obtained results suggested the preferential deposition of the self-aggregated IgA composed of hypoglycosylated IgA1 and co-deposition of IgG, IgM and C3 in the glomeruli in an IgA nephropathy patient.  相似文献   

11.
12.
MitoTracker Green (MTG) is a mitochondrial-selective fluorescent label commonly used in confocal microscopy and flow cytometry. It is expected that this dye selectively accumulates in the mitochondrial matrix where it covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Here we demonstrate that MTG can be used as a protein labeling reagent that is compatible with a subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Although the MTG-labeled proteins and MTG do not seem to electrophoretically separate, an enhancement in fluorescence intensity of the product indicates that only proteins with free thiol groups are capable of reacting with MTG. In addition we propose that MTG is a partially selective label towards some mitochondrial proteins. This selectivity stems from the high MTG concentration in the mitochondrial matrix that favors alkylation of the available thiol groups in this subcellular compartment. To that effect we treated mitochondria-enriched fractions that had been prepared by differential centrifugation of an NS-1 cell lysate. This fraction was solubilized with an SDS-containing buffer and analyzed by CE-LIF. The presence of a band with fluorescence stronger than MTG alone also indicated the presence of an MTG-protein product. Confirming that MTG is labeling mitochondrial proteins was done by treating the solubilized mitochondrial fraction with 5-furoylquinoline-3-carboxaldehyde (FQ), a fluorogenic reagent that reacts with primary amino groups, and analysis by CE-LIF using two separate detection channels: 520 nm for MTG-labeled species and 635 nm for FQ-labeled species. In addition, these results indicate that MTG labels only a subset of proteins in the mitochondria-enriched fraction.  相似文献   

13.
In the current paper, we report the development of a new capillary electrophoresis method using pre-column derivatization and laser-induced fluorescence detection for the determination of ephedrine and amphetamine drugs. Our new method allows for the identification and quantification of six commonly used illicit drugs namely pseudoephedrine, ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethylamphetamine, respectively, as well as propafenone (internal standard). Following derivatization with fluorescein isothiocyanate, a total of six amphetamine drugs and the internal standard could readily be separated using a fused-silica 75 micromID x 60 cm length (effective length: 50.2 cm) capillary column. The mobile phase consisted of buffer containing 20mM borate (pH 12, adjusted with sodium hydroxide). Samples were injected in pressure mode with the capillary being operated at 25kV/25 degrees C, and the detection of the derivatized compounds was sought using a laser-induced fluorescence (LIF) detector (lambda(ex)=488 nm and lambda(em)=520 nm), with a run-time of 20 min. The current method was validated with regard to precision (relative standard deviation, RSD), accuracy, sensitivity, linear range, limit of detection (LOD) and limit of quantification (LOQ). In human blood and urine samples, detection limits were 0.2 ngmL(-1), and the linear range of the calibration curves was 0.5-100 ngmL(-1). The intra-day and inter-day precisions were both less than 13.22%.  相似文献   

14.
The protein analysis of structural tissues is typically highly problematic. Amniotic membrane displays unique wound healing and anti-scarring properties; however, little is known concerning its active protein content. The structural nature of amniotic membrane necessitated development and extensive optimisation of the entire two-dimensional (2-D) workflow. Proteins were extracted using powerful solubilisation buffers and analysis carried out using 2-D electrophoresis followed by mass spectrometry (MS) identification. Preservation and processing resulted in prefractionation of soluble from structural and membrane-associated proteins. Enhanced protein solubility was achieved by cysteine blocking using both N,N-dimethylacrylamide (DMA) alkylation and bis(2-hydroxyethyl) disulphide (HED); an alternative procedure for the effective application of HED is demonstrated. The benefits of precipitation and cup-loading versus in-gel rehydration were also assessed, with procedures for the employment of HED with the latter described. Following optimisation, a representative sample 21 proteins were identified from amniotic membrane using MS verify procedures were MS-compatible. Our results demonstrate that techniques for the reproducible separation of proteins from a proteinaceous structural tissue have been optimised. Briefly, proteins are extracted using a thiourea/urea extraction buffer containing carrier ampholytes, dithiothreitol (DTT), and 3-(cyclohexylamino)-1-propanesulfonic acid (CHAPS). After DMA alkylation, proteins were precipitated (using the 2-D clean-up kit from Amersham Biosciences) and resolubilised in extraction buffer containing a lower concentration of DTT. Samples were either cup-loaded onto rehydrated HED-containing strips or rebuffered into HED-containing buffer followed by in-gel rehydration.  相似文献   

15.
Absence of a convenient, direct enzyme assay for detecting phenylketonuria (PKU) heterozygotes has resulted in continued effort to develop an accurate and reliable procedure to discriminate the heterozygous individual from the homozygous normal. Our study compares two statistical procedures that combine the semifasting plasma phenylalanine and tyrosine concentrations with the individuals' prior probability of being a heterozygous carrier in order to discriminate carriers from noncarriers. The results of this comparison indicate that the quadratic discriminant function is superior to the linear discriminant function as a method of carrier testing both in theory and in practice. An interactive computer system is described that facilitates the clinical utilization of the quadratic discriminant function.  相似文献   

16.
Summary The major endosperm proteins in a range of genotypes of hexaploid wheat have been fractionated by two-dimensional electrophoresis. The genotypes included nine varieties and forty four intervarietal substitution lines in which chromosomes 1A, 1B, 1D, 6A, 6B or 6D from eight of the varieties have been introduced one at a time into a common genetic background. The appearance of different protein subunits was often correlated with a chromosome substitution. This showed that many of the genes for the high molecular weight protein subunits (molecular weight range 55,000 to 140,000 determined by SDS polyacrylamide gel electrophoresis) are specified by chromosomes 1A, 1B and 1D while many of the lower molecular weight subunits (molecular weight range 30,000 to 45,000) are specified by chromosomes 6A, 6B and 6D. The different protein subunits correlated with chromosome substitution could not always be recognised in the varietal source of the substituted chromosome. The different subunits specified by homologous chromosomes in different wheat varieties may differ in isoelectric point and/or molecular weight.  相似文献   

17.
Two-dimensional electrophoresis of proteins and protein subunits, employing isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate in the second, yields the highest resolutions currently available. In this paper separations in the second dimension are considered (the so called DALT system). Methods for multiple-parallel casting of gradient gels in slab gel holders are described. The problem of electrical isolation of the ends of the slabs together with continuous cooling of both surfaces of the slab gel holder along their entire length has been achieved by running the gels in a horizontal direction in a three-compartment tank with the holders inserted in insulating septa. In the system described, 10 slabs are run simultaneously. This, however, is not the upper limit of the number of slabs which can be conveniently run in parallel.  相似文献   

18.
The analysis of protein pharmaceuticals currently involves a complex series of chromatographic, electrophoretic, spectroscopic, immunological and biological measurements to unequivocally establish their identity, purity and integrity. In this review, I briefly consider the possibility that at least the functional identity and integrity of a protein drug might be established by either a single analysis involving X-ray diffraction, NMR or mass spectrometry, or by a chromatographically based multi-detector system in which a number of critical parameters are essentially simultaneously determined. The use of a protein standard to obtain comparative measurements and new advances in the technology of each of these methods is emphasized. A current major obstacle to the implementation of these approaches is the frequent microheterogeneity of protein preparations. The evolution of biological assays into measurements examining more defined intracellular signal transduction events or based on novel biosensors as well as the analysis of vaccines is also briefly discussed.  相似文献   

19.
The present study describes the simultaneous determination of four drugs, two local anaesthetics (lidocaine and bupivacaine) and two opium alkaloids (noscapine and papaverine) by capillary zone electrophoresis (CZE) with solid-phase extraction (SPE) procedure using Oasis HLB cartridges. Their recoveries ranged from 81 to 107% at the target concentrations of 2.0, 5.0 and 8.0 μg mL?1 in spiked urine samples. Coefficients of variation of the recoveries ranged from 2.1 to 11.3% at these concentrations. The quantitation limits of the method were approximately 300 ng mL?1 for the different compounds studied. The assay is very specific for these compounds and requires a short sample preparation procedure prior to the electrophoretic analysis.  相似文献   

20.
Glycosyltransferases are key enzymes in glycoconjugate biosynthesis, which make them important targets for biomedical research. Among the different methodologies developed to analyze glycosyltransferase activities, fluorophore-assisted capillary electrophoresis (FACE) emerges as a powerful technique in carbohydrate analysis. Its application to monitor glycosyltransferase activity has been limited to reactions with derivatized sugars as acceptor substrates in which a charged fluorophore/chromophore must be introduced, thus requiring tedious preparative synthesis and purification for each single acceptor substrate. Here we describe a novel and general glycosyltransferase assay based on FACE using underivatized acceptor substrates. Enzyme activity is monitored by a discontinuous assay with postreaction derivatization by reductive amination with 8-aminonaphthalene-1,3,6-trisulfonic acid. The reaction mixture is directly analyzed by HPCE (high-performance capillary electrophoresis) under inverted electroosmotic conditions at pH 2.5 and 30 degrees C. After method validation, it was applied to the kinetic characterization of an alpha-1,3-galactosyltransferase, the enzyme responsible for the biosynthesis of alphaGal epitope involved in the hyperacute rejection in xenotransplantation. The absence of a label on the acceptor during the GT reaction avoids any interference of the label with the enzyme, and the postreaction derivatization does not require any purification step.  相似文献   

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