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The use of a phenylalanine (Phe) functionalized tentacle-type polymer coated capillary column for protein separation by open tubular capillary electrochromatography (OTCEC) was demonstrated in this work. The tentacle-type stationary phase was prepared from silanized fused-silica capillaries of 50 microm I.D. by glycidyl methacrylate graft polymerization and subsequent Phe functionalization. Due to the amphoteric functional groups of the Phe bonded on the tentacle-type polymer stationary phase, protein separation in the prepared column can be performed under both cathodic and anodic electroosmotic flow (EOF) by varying the pH values of the mobile phase. Model proteins including ribonuclease A (RNase A), myoglobin, transferrin, insulin were baseline separated under cathodic EOF with a mobile phase of pH 8.8. Comparison between the separation result of the four proteins under conditions of OTCEC and capillary zone electrophoresis indicates that the migration behavior of the four proteins in the prepared column was the result of the interplay of chromatographic retention and electrophoretic migration. Besides, three basic proteins including RNase A, cytochrome c (Cyt-c) and lysozyme (Lys) were fully resolved under anodic EOF with an acidic running buffer (pH 2.5). The elution order was the same as the isoelectric point values of the proteins (RNase A

The behavior of chromatographic columns packed with resins containing both weak and strong cation-exchange groups is investigated in order to obtain protein separations by means of internally generated pH gradients in response to step changes in buffer composition. A local equilibrium model is developed to predict pH transitions using non-adsorbed buffers, i.e. containing neutral and negatively charged buffering species, based exclusively on the resin titration curve. In agreement with experimental results, the model predicts practical, fairly linear gradients between pH 5 and 7, which are formed using suitable mixtures of acetate and phosphate buffers. The separation of mixtures of ovalbumin, albumin, and transferrin is used as a model system, but, unlike most previous work, we consider preparative conditions. Near baseline resolution is obtained with protein loads as high as 10mg/mL and mobile phase velocities at high as 460 cm/h using porous, 70-microm diameter particles. The peaks obtained with this approach are much sharper than could be obtained isocratically or using externally generated, unretained gradients as a result of the peak compression caused by the axial pH gradient formed along the column. Moreover, separation is obtained at very low ionic strengths (2-3 mS/cm). The effects of flow velocity, mobile phase composition, time of injection, and protein load on retention and elution pH are investigated systematically demonstrating a range of ways in which the separation can be controlled and optimized.

Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electro-osmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, alpha1-antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10(-9) m2V(-1)s(-1). They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and alpha1-antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics.

High-speed counter-current chromatography (HSCCC) is a continuous liquid-liquid partition chromatography, with remarkable advantages of high separation efficiency and no adsorption or denaturation by solid phase. The retention of stationary phase and the separation of proteins in polyethylene glycol 1000 (PEG1000)-phosphate aqueous two-phase system (ATPs) were studied with a multi-column high speed-counter-current chromatograph. The flow direction and speed of the mobile phase, and the rotation direction and speed of the apparatus showed different effects on the retention of the stationary phase, which reached the maximum at 33.3% with a flow rate of 0.6 mL/min and a rotation speed of 900 r/min in 14.0% PEG1000-16.0% phosphate ATPs. Distinct differences in partition coefficients among cytochrome C, lysozyme and hemoglobin were found at pH 9.2 and these three proteins were successfully separated in 14.0% PEG1000-16.0% phosphate ATPs at pH 9.2 by HSCCC with the apparatus rotating at 850 r/min and the mobile phase flow rate of 1.0 mL/min. The major protein components in hen egg white, including ovaltransferrin, ovalbumin and lysozyme also show distinct differences of partition coefficients in PEG1000-phosphate ATPs at pH 9.2. Ovalbumin and lysozyme were successfully purified to homogeneity and ovaltransferrin to ca 60% purity from the hen egg white sample with yields over 90% in 15.0% PEG1000-17.0% phosphate ATPs at pH 9.2 with the apparatus rotating at 850 r/min and mobile phase flow rate of 1.0 mL/min.

The use of liquid chromatography in the separation and determination of metal-binding proteins is reviewed. Advantages and drawbacks of different chromatographic techniques based on various principles: size exclusion, ion exchange (cationic and ionic), reversed phase and affinity, are presented and discussed. The topic "metal-binding proteins" is considered and presented from two different points of view. The first one regards metal speciation in biological samples (serum and blood). In metal speciation studies, the exact identity of the protein to which the metal is bound often remains unknown. The second point of view is that, frequently, the interest of analyzing metal-binding proteins is not related anymore to the metallic fraction of the protein, but to other chemical structures attached to the protein, such as carbohydrates, which indirectly determine how good the function of the protein is. In this review, special attention is paid to studies dealing with the glycosylation of transferrin, and with the glycated isoform of haemoglobin.

Indocyanine green (ICG)--a negatively charged, polymethine dye--can interact noncovalently with proteins to form fluorescent complexes, with excitation and emission maxima near 780 and 820 nm, respectively. This behavior was realized utilizing either a 100 mM phosphate buffer or a 25 mM citric acid buffer, both at pH 3.1. The behavior of ICG under these conditions, termed pseudofluorogenic, rendered the dye suitable for use as a label for protein determination in capillary electrophoresis with diode laser-induced fluorescence detection (CE-LIF). To this end, pseudofluorogenic ICG was used both as an on-column label for human serum albumin (HSA) and as a precolumn label for a model mixture of proteins, including ribonuclease A, transferrin, and cytochrome c. These ICG-labeled proteins were successfully resolved in less than 11 min, with no interference from excess, unbound dye.

We have developed a density gradient electrophoresis device (DGE) and used it for the preparative separation of various endocytic organelles that are hard to separate by other means. Our separation by DGE of late endosomal vesicles, recycling vesicles, early endosomes and plasma membranes is unmatched. Using the same DGE device, we performed preparative high-resolution rate zonal separation of proteins using amphoteric buffers as originally described by Bier (Electrophoresis 1993, 14, 1011-1018). Isoforms of bovine beta-lactoglobulin, human apo-transferrin, and bovine erythrocyte carbonic anhydrase that have isoelectric points within 0.8 pH units were readily separated even in the absence of nonionic detergents. The DGE apparatus is inexpensive and has unique separation abilities for vesicles and proteins.

In this report we describe a novel approach to the mass spectrometric analysis of whole proteins from gels. The strategy consists of three components: conventional SDS-PAGE gels, reversible negative staining procedures, and passive elution of proteins from gels followed by mass spectrometric analysis. Protein bands are excised from SDS-PAGE gels, destained, and extracted. For gel loadings > or = 25 pmol of soluble protein, the proteins can be directly extracted into a solution consisting of formic acid/water/2-propanol. The recovered protein is suitable for matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization mass spectrometric analysis. For gel loadings < 25 pmol protein, the mass spectrometric response, using the direct extraction procedure, drops off sharply, an outcome that is attributed to protein recovery losses. To offset the protein losses, the extraction procedure is slightly modified by performing the passive extraction of the gel with a saturated MALDI matrix solution. During the extraction period, the matrix is allowed to crystallize, forming a suspension in solution. Protein that elutes from the gel has a chance to cocrystallize with the matrix that can be retrieved for MALDI-MS analysis. This method of "capturing" eluted protein into matrix crystals is sensitive to 1 pmol of recombinant mouse leptin protein (16 kDa) loaded onto SDS-PAGE gels and can be used for proteins as large as 70 kDa. Our strategy has particular application to the characterization of endogenous forms of mature proteins from SDS-PAGE gels.

High effective specific activity N-succinimidyl 4-[18F](fluoromethyl)benzoate was prepared using a reversed phase HPLC procedure. Reversed phase HPLC removed several additional impurities not removed by normal phase HPLC, thereby increasing the effective specific activity. Small amount (< 100 micrograms) of sensitive proteins such as erythropoietin can be labeled with this reagent in high yield without aggregation.

The performance of capillary gel-sodium dodecyl sulfate (SDS) electrophoresis for molecular-mass-dependent separation of proteins has been examined employing cross-linked polyacrylamide gels and untreated fused-silica capillaries of short length. An apparatus for capillary electrophoresis has been constructed, combining a UV detector equipped with a capillary holder, a power supply, and a micromanipulator for sample loading. The apparatus is capable of accommodating gel capillaries of different lengths and can be operated with two methods of sample loading: manual injection on top of the gel or electrokinetic injection. Standard proteins have been separated according to their molecular mass (ranging from 17000 to 116000) within 30 min, employing gel capillaries of effective lengths shorter than 10 cm and polyacrylamide gels ranging from 2.4 to 4.8%T (5%C). The results confirmed that gel capillaries of effective length less than 10 cm can be used for protein size-separation by SDS electrophoresis, with much higher performance than is achieved in rod gel-SDS electrophoresis.

In view of the evidence showing that germ cells regulate Sertoli cell (SC) function, the aim of this study was to examine if germ cell (GC)-conditioned media contained multiple biological factors that affect SC secretory functions. Total GC were isolated from adult rat testes. Pachytene spermatocytes (SPC) and early spermatids (SPT) were enriched to about 90% pure by centrifugal elutriation. GC, SPC, and SPT were cultured in serum-free medium for 20 hours with a viability greater than 95%. Conditioned media derived from these cells were fractionated by anion-exchange high-performance liquid chromatography (HPLC). An aliquot from each of these fractions was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were visualized by silver staining. The patterns of protein staining using media from GC, SPC, and SPT were similar. Bioassays of these column fractions on SC showed that the transferrin stimulatory, the testins inhibitory, and the clusterin inhibitory activities were eluted from the anion-exchange HPLC column in overlapping fractions. To determine whether these activities were confined to one or several molecules, further fractionations were performed. Eleven liters of GC-conditioned medium were fractionated by sequential HPLC using anion-exchange, gel permeation, and reversed-phase columns. Throughout the entire fractionation scheme, the HPLC fractions were bioassayed using primary SC-enriched cultures prepared from 20-day-old rats by incubating SC with aliquots of these fractions for 24 hours to monitor their effects on SC secretory function. The concentrations of transferrin, clusterin, and testins were quantified by specific radioimmunoassays. These studies showed that the transferrin stimulatory activity can be fractionated into four peaks (I, IIa, IIb, and IIc); clusterin inhibitory activity into three peaks (A, B, and C); and testins inhibitory activity into two peaks (1 and 2). Some of these bioactivities were eluted in overlapping fractions such as I and B, IIb and 1, and IIc and 2, whereas A, C, and IIa were not associated with any other assayed activities. In summary, additional GC modulators of SC function were identified for the first time, these include clusterin and testins inhibitors. The previously identified transferrin stimulatory activity was also resolved into multiple molecular forms.

The influence of sodium chloride concentration and the pH of the mobile phase on the distribution coefficient of proteins with different pI values was studied on Sephacryl S-200 HR. The non-size-related behaviour of this gel filtration packing is mainly attributed to small amounts of groups that are negatively charged within the pH range investigated (4.2-10.0). These anionic groups on the packing gave rise to ion-exchange or ion-exclusion interactions depending on the charge characteristics of the protein. Hydrophobic interactions at high ionic strength and intramolecular electrostatic repulsive interactions at low ionic strength were also observed for some proteins. The chemical stability of Sephacryl S-200 HR was studied by comparing the chromatographic results with Sephacryl S-200 HR that had been treated in acidic or basic solutions with those with fresh Sephacryl. After Sephacryl S-200 HR had been stored for 2 weeks in 0.10 M sodium hydroxide the chromatographic results at low ionic strengths clearly showed that groups that are positively charged at pH 4.2 had been formed. However, storage for 2 weeks in 0.01 M hydrochloric acid did not change the chromatographic behaviour of the proteins from that observed when injected on fresh Sephacryl S-200 HR.

Simple improvements of the crossed immunoelectrophoresis method are described. A trap-gel was prepared with a small quantity of monospecific antiserum and was submitted to preelectrophoresis. It blocked, during the first dimension, an individual protein as a rocket. The corresponding peak was reduced or disappeared in the second dimension. Identification of precipitation peaks in human or Macacus cynomolgus bronchoalveolar lavage fluids is illustrated and unequivocal recognition of some antigens is shown.