Rapid isoelectric focusing of proteins in hydrolytically stable capillaries.

Three new types of capillary coatings for capillary isoelectric focusing that avoid siloxane chemistry, resulting in hydrolytically stable coatings, are described and tested: phenyl-silica, acrylamide-reacted vinyl-silica, and pure PTFE. Capillaries of these three types were compared using standard proteins and a biological mixture of proteins similar to what might be encountered in actual use. Of these, the acrylamide-coated capillary produced the highest-quality results. In contrast to capillaries prepared using siloxane reactions, the capillaries described herein exhibited greatly enhanced stability at high pH.     

Fast isoelectric focusing of milk proteins on small ultrathin polyacrylamide gels containing urea

The preparation of 0.45 mm thin polyacrylamide gels, containing urea, for horizontal micro isoelectric focusing of milk proteins with PhastSystem is described. Isoelectric focusing in the small gels, stained either with Coomassie Brilliant Blue R-250 or with the more sensitive silver stain, affords a fast and sensitive procedure for an analysis of milk and cheese proteins. The procedure can be effectively exploited in detecting adulteration in ovine cheese with bovine milk.     

Determination of the isoelectric point of proteins by capillary isoelectric focusing

Different ways of determining isoelectric points (pI) of proteins in capillary isoelectric focusing are reviewed here. Due to the impossibility of direct pH measurements in the liquid phase, such assessments have to rely on the use of pI markers. Different types of pI markers have been described: dyes, fluorescently labelled peptides, sets of proteins of known pI values. It appears that, perhaps, the best system is a set of 16 synthetic peptides, trimers to hexamers, made to contain each a Trp residue for easy detection at 280 nm. By a careful blend of acidic (Asp, Glu), mildly basic, with pK around neutrality (His), and basic (Lys, Arg) amino acids, it is possible to obtain a series of pI markers with pI values quite evenly distributed along the pH scale, possessing good buffering capacity and conductivity around their pI values and thus focusing as sharp peaks. Another approach to pI determination is the monitoring of the current during mobilization: this allows, with the aid of known pI markers, to calibrate the system with a pI/current graph. Pitfalls and common errors in pI determinations are reviewed here and guidelines given for minimizing such errors in pI estimation.     

A new sample applicator for isoelectric focusing in horizontal polyacrylamide gels

A new sample applicator for horizontal flat gels is described. The applicator is practically safe against contamination from adjacent samples and can be used for all types of electrophoretic separations including a concentration step for either the sample (i.e. disc electrophoresis) or the separated zones (i.e. isoelectric focusing). The applicator is a piece of flat glass with 26 or 51 parallel 2 mm wide grooves, drilled at distances of 9 or 4.5 mm. Samples, maximally 25 or 50, are applied to the areas between the grooves. By inverting the applicator, the samples are brought into close vicinity to the gel surface and the pendant droplets expand by capillary attraction into the slits between the glass and gel with resultant even distribution across the lanes of 2.5 or 7 mm width. The applicator can be used for separations with and without protection of the electrophoretic setup by paraffin oil and allows for fast multiple handling of samples by means of appropriate syringes and microtiter plates.     

Preparative isoelectric focusing of reduced wheat gluten proteins

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.     

Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients.

Isoelectric focusing in immobilized pH gradients (IEF-IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion-exchange and reversed-phase chromatography proved to be homogeneous with regard to its isoelectric point (pI). In addition, the theoretical pI, calculated on the basis of the primary structure, corresponded precisely to the measured pI of 4.30. IEF-IPG was further employed to follow the stability of recombinant hirudin at pH 9, indicating that deamidation occurred under these conditions. A variant of recombinant human alpha 1-antitrypsin (AAT) (389 amino acids, one cysteine residue) expressed in Escherichia coli and purified by anion-exchange, metal chelate and hydrophobic-interaction chromatography appeared to be homogeneous by polyacrylamide gel electrophoresis under reducing and denaturing conditions as well as by various high performance liquid chromatography methods. However, some heterogeneity was detected by IEF-IPG between pH 5-6. The measured pI values of 5.43-5.58 were slightly lower than the calculated pI based on the primary structure (5.72). This indicated deamidations of Asn or Gln residues. A recombinant Schistosoma mansoni parasite antigen, p28 (210 amino acids, one cysteine residue) obtained after intracellular expression in Saccharomyces cerevisiae and affinity purification on glutathione agarose was analyzed by IEF-IPG in a pH 7.3-8.3 gradient. It appeared to be heterogeneous with regard to its pI, with the major component having a pI of 7.81 compared to the calculated value of 7.17.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemiluminescence detection of heme proteins separated by capillary isoelectric focusing.

Chemiluminescence detection was combined with capillary isoelectric focusing to perform protein analysis with high sensitivity. Luminol-H2O2 chemiluminescence was utilized, and heme proteins such as cytochrome c, myoglobin and peroxidase were analyzed. The proteins were focused by use of Pharmalyte 3-10 as ampholytes. Hydroxypropylmethyl-cellulose was added to the sample solution in order to easily reduce protein interactions with the capillary wall as well as the electroendoosmotic flow. The focused proteins were transported by salt mobilization to chemiluminescence detection cell equipped with an optical fiber. The present method showed significantly high sensitivity and wide dynamic range; the detection limit for cytochrome c was 6 x 10(-9) M and the linear dynamic range was greater than two-orders of magnitude (up to 2 x 10(-6) M).     

Rehydratable agarose gels: application to isoelectric focusing in 9 molar urea

A method is described for the preparation of rehydratable agarose gels, with specific application to the direct incorporation of 9 M urea and carrier ampholytes into rehydratable agarose gels for use in isoelectric focusing. After drying the agarose gel containing an uncharged linear polyacrylamide, one gel volume of a 9 M urea-carrier ampholyte solution is absorbed directly into the gel in 60 min, eliminating equilibration or dialysis of the gel in larger volumes of the 9 M urea-carrier ampholyte solution. Proteins with a molecular mass of 970,000 Da can be separated by isoelectric focusing in these rehydratable gels. The focused proteins can then be quantitatively transferred to nitrocellulose in less than 10 min, and any immunostaining procedure can be used to probe the blotted proteins. These agarose gels are easy to make, they rehydrate rapidly and they can be used in applications other than isoelectric focusing.     

Identification of gram-negative bacteria by isoelectric focusing of soluble cellular proteins.

Strains of Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis, isolated from clinical specimens were disrupted by repeated freezing in liquid nitrogen and thawing at room temperature. The samples were separated by isoelectric focusing using polyacrylamide gels. The resulting protein patterns showed clear differences between the three species and made identification possible.     

Nonamphoteric isoelectric focusing: II. Stability of borate-glycerol pH gradients in recycling isoelectric focusing

By complexing polyols with borate in recycling isoelectric focusing and by varying the ratio of polyol to borate over the useful pH range of 4.0-6.0, it is possible to control pH. Twelve solutions of 0.1 M boric acid and varying glycerol concentration were used to vary pH in a twelve-compartment commercial recycling isoelectric focusing (RIEF) system. Various concentrations of boric acid were tested as anolyte, and various Tris(hydroxymethylamino)methane-borate buffer systems were tested as catholyte. Electroosmosis, hydrogen ion flow, and fluid balancing were characterized in two glycerol gradients; one was maintained at 0.06 pH/fraction and the other at 0.12 pH/fraction. In the latter case, ovalbumin (pI4.70) migrated to the pH 4.61 and 4.72 compartments. It is concluded that the borate-glycerol system can be adequately stabilized in RIEF for isoelectric purification of certain proteins.     

Immobilized pH gradient isoelectric focusing of cerebrospinal fluid proteins

Isoelectric focusing in immobilized pH gradients, supplemented with 0.5% w/v carrier ampholytes was applied for studies of native proteins, especially immunoglobulin G, in cerebrospinal fluid and serum. All 72 paired samples were run on pH 4-10 gels; 25 of them were also examined in pH 7-10 gels. Silver staining and nitrocellulose blotting with amplified immunoperoxidase detection of immunoglobulin G were used for protein visualization. Intrathecally produced immunoglobulin G was resolved into sharply focused, straight and easily identifiable fractions. The pH gradients were stable and the inter-gel reproducibilities of individual immunoglobulin G patterns were good.     

Rapid high-performance isoelectric focusing of monoclonal antibodies in uncoated fused-silica capillaries

Rapid (<5 min) high-performance isoelectric focusing was performed in uncoated fused-silica capillaries to resolve isoforms of monoclonal antibodies and to determine their isoelectric points (pI). The methodology involved the use of a 32 cm (effective length 9 cm)×50 μm I.D. uncoated capillary. (Hydroxypropyl)methyl cellulose was used as an additive to suppress analyte–wall interaction and to precisely control electroosmotic flow so that focusing and mobilization of focused zones past detector occur simultaneously. Urea was included in the separation medium to solubilize the antibodies that precipitated at their point of focusing. The methods with and without urea used ampholytes pH 5–8 to generate a demonstrable linear gradient between pH 5.4 and pH 7.2, based on the separation of various protein standards. Reproducibility [<2% (R.S.D.)] of the migration times (corresponding to the detectable isoforms of the antibodies) was obtained by using two sets of reagents and capillaries on three consecutive days. pI values determined from day-to-day with a reference standard were shown to vary by only 0.01 pH unit. The described capillary isoelectric focusing methods provided a rapid, simple and reproducible way of monitoring micro-heterogeneity and pI of the murine monoclonal antibodies investigated.     

Miniaturization of capillary isoelectric focusing.

Miniaturization of whole-column imaging capillary isoelectric focusing (CIEF) is discussed. A 1.2 cm capillary was used as a separation column for CIEF. The experimental results for the analysis of two pI markers and the protein myoglobin showed that good CIEF separation results could be obtained. Secondly, a light-emitting diode (LED) was used as the light source for the whole-column absorbance imaging detection. The focusing of both the pI markers and myoglobin were observed with the LED light source. The whole-column imaging CIEF instrument was simplified and miniaturized by the use of the LED. Further developments are also discussed.     

Effect of salt on the performance of immobilized pH gradient isoelectric focusing gels

The effect of salt and buffer ions in the sample or in an immobilized pH gradient (IPG) on sample entry into the gel and on the final focused pattern are presented. During the initial phase of electrofocusing, ions present in the gel, either as counter ions to the immobilized charge groups of the IPG gel or added to the gel matrix during the rehydration process, are transported toward the electrodes. For ions present at a concentration exceeding approximately 1 mM the transport can be followed by the refractile line marking the trailing edge of an ion-containing zone. Gradual sample entry may be achieved by applying the sample at a site (near the anode or cathode) opposite to that from which the sharpest refractile line, marking the ion present in the highest concentration, approaches the sample. Additionally, lateral band spreading of the sample is avoided. Thus, sample applied at the cathode for IPG gels rehydrated with 1-2 mM Tris base, or at the anode for gels rehydrated with 1-2 mM acetic acid or sodium acetate, enters the gel matrix gradually without lateral band spreading. In contrast, sample applied at the anode, for Tris-containing gels, or at the cathode, for acetate-containing gels, enters rapidly in a sharp zone when the refractile line reaches the sample zone. This results in a high local protein concentration in the zone immediately behind the boundary with lateral band spreading.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimation of isoelectric points of human plasma proteins employing capillary isoelectric focusing and peptide isoelectric point markers

Synthetic UV-detectable peptide pI markers were used to estimate isoelectric point (pI) values of proteins separated by capillary isoelectric focusing (CIEF) followed by cathodic mobilization in the absence of denaturing agents. The pI calculation and quantitative analysis of purified proteins showed the feasibility of these peptides as pI markers and internal standards in CIEF separation of proteins. Estimation of pI values of major proteins in human plasma was performed using the peptide pI markers, and the values were compared with those previously obtained by gel isoelectric focusing (IEF). Sera of immunoglobulin G (IgG) myeloma patients, which showed characteristic peaks of myeloma IgG in their CIEF patterns, were also subjected to the analysis and the pI values of the myeloma proteins have been estimated.     

Effects of increased voltage on resolution in preparative isoelectric focusing of myoglobin varia

IEF is a powerful technique which separates proteins and other amphoteric solutes in a pH gradient according to their pI's. The current work evaluates the effect on resolution of increasing electric fields in a novel preparative, vortex-stabilized electrophoresis device. In shallow gradients spanning one pH unit, the variants of myoglobin were separated at applied voltages from 10 to 15 kV. Digital imaging of these separations indicated a 20% reduction in bandwidth and a 60% increase in resolution as the electric field strength is varied across this range. These results were confirmed by IEF-PAGE and ion-exchange chromatography.     

Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250

An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.     

Haptoglobin subtyping by isoelectric focusing in miniaturized polyacrylamide gels rehydrated in presence of 2-mercaptoethanol

A fast isoelectric focusing method for routine haptoglobin (Hp) subtyping is presented. This method is based on isoelectric focusing, under reducting conditions, of neuraminidase-treated plasma samples by using dry miniaturized (interelectrode distance: 55 mm) polyacrylamide gel, rehydrated in presence of 2-mercaptoethanol and a mixture of pharmalyte carrier ampholytes (pH 4-6.5 and pH 6-8) followed by immunoblotting. The presence of 2-mercaptoethanol in the gel prevented refolding of the Hp alpha and Hp beta chains during focusing, making it possible to obtain a sharp Hp band pattern with a clear separation of the different Hp alpha allelic products (1S, 1F, 2FS, 2SS and 2FF). A population study carried out with 250 unrelated individuals living in Central Spain is also presented.