Immobiline Gels Research Articles

Single step separation and concentration of biomarker proteins using agarose based miniaturized isoelectric gates for point of care diagnostics

Bedside diagnostics using protein biomarkers requires rapid concentration, isolation and measurement of these specific biomarkers from a complex matrix in a low cost and portable manner. Traditional separation using gel electrophoresis separates the sample into all its constituent elements and requires high voltages and/or long runtime which is often unnecessary for diagnostics where only a specific biomarker needs to be quantified. Digital isoelectric trapping can be used to isolate and concentrate proteins but requires UV cured immobiline gels that have defined pH values and are not tunable to target specific proteins. Here, we have developed miniaturized isoelectric gates to separate, concentrate and quantify a targeted biomarker based on its isoelectric point, from a complex matrix in under 20 min. We designed specific isoelectric gates to concentrate and quantify bovine serum albumin (BSA) at a concentration of 1−5 mg/mL with a peak concentration of over 300 mg/mL, while maintaining its solubility. Next, we have demonstrated isolation of human protein C from a mixture with 1000-fold higher concentration of BSA with no additional processing. Finally, we have demonstrated rapid (< 20 min) separation, concentration and quantification of ovomucoid in a fresh hen egg using dual isoelectric gates at pH 3.9 and 4.3 to trap ovomucoid with an isoelectric point of 4.1 using a low applied voltage of only 15 V. By using low cost agarose gels instead of expensive immobilines, this device reduced the cost and complexity of separation. The combination of low-cost, tunability, fast-runtime, low-operating voltage makes this technique attractive for use in point-of-care biomarker detection without specific antibody tags.

Analysis of protein species differentiation among mycobacterial low-Mr-secreted proteins by narrow pH range Immobiline gel 2-DE-MALDI-MS

Secreted proteins of bacteria are preferentially capable of interacting with host cells and are therefore of special biological and medical interest. Narrow pH range 2-DE and MALDI-TOFTOF-MS combine high-resolution protein separation with highly sensitive identification of proteins. Secreted proteins of Mycobacterium tuberculosis were separated at the protein species level, distinguishing different protein species of one protein. We focused on the pI range 4.0-4.7 and the Mr range 6-20kDa of the 2-DE pattern. Out of 128 analyzed spots, 121 were identified resulting in 33 different proteins with 277 different protein species, accumulating in a mean of 8.4 protein species per protein. Overrepresentation was found for the protein classes "virulence, detoxification, adaption", "information pathways", "cell wall and cell processes" and "intermediary metabolism and respiration". Thus far, 15 protein species of the ESX-1 family are characterized with 100% sequence coverage. More automated 2-DE procedures and more sensitive identification techniques are required for complete characterization of all of the protein species even in highly enriched samples, such as culture filtrates. Only then the functional level of proteomics will be achieved and potential biomarkers can be postulated at the molecular level. Proteomics is dominated by bottom-up approaches largely ignoring protein speciation. A prerequisite to reach the protein species level is to obtain 100% sequence coverage, which is a major challenge in proteomics. Here we show complete sequence information with a 2-DE-MS approach for 15 protein species. Acetylation of the N-terminus of ESAT-6 inhibits interaction with CFP-10, with direct consequences for pathogen-host interaction. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Laser microdissection and two-dimensional difference gel electrophoresis with alkaline isoelectric point immobiline gel reveals proteomic intra-tumor heterogeneity in colorectal cancer

To approach heterogeneity of tumor tissues, we examined proteomes of normal and tumor tissues from colorectal cancer patients by laser microdissection and 2D-DIGE with saturation dye and alkaline range IPG gel. By distinguishing the tumor cells based on tissue localization, we found that a range of protein spots showed different intensity between normal and diseased tissues. Subsequent mass spectrometric protein identification identified the proteins for protein spots. We found that the protein spots from identical proteins showed the different tissue localization in colorectal cancer. These results highlight the potential utilities of laser microdissection, 2D-DIGE, and immobilized pH gradient gel with alkaline range.

Polar Electrophoresis: Shape of Two-Dimensional Maps Is as Important as Size

The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of “isobaric” spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis.

Proteomic Analysis of Erythrocyte Membranes by Soft Immobiline Gels Combined with Differential Protein Extraction

Improvements in proteome analysis of erythrocyte membrane proteins by two-dimensional electrophoresis are here reported. In particular, a differential extraction procedure was set up allowing separation of integral membrane proteins from peripheral species. Moreover, the use of dilute Immobiline gels (down to as low as 3% T matrix) permitted a better penetration and transfer inside the gel of proteins with large M(r). These protocol modifications, combined with sample delipidation and alkylation prior to electrophoresis, which prevented generation of homo- and hetero-oligomers following disulfide scrambling phenomena, allowed the display of more than 500 spots in the pI/M(r) plane. Among those, noteworthy was the presence of high levels of filamentous proteins, such as alpha-spectrin and ankyrins, or integral membrane proteins, such as band 3, band 4.1 and 4.2, not displayed or barely present in other maps exploiting immobilized pH gradients in the first dimension. Accordingly, our results show that this 2D mapping technique is a valuable tool in exploring pathologies related to genetic defects associated to membrane proteins.

Parallel isoelectric focusing chip.

Fast isoelectric focusing (IEF) is becoming a key method in modern protein analysis. We report here the theory and experimental results of new parallel isoelectric devices (PID) for fast IEF. The main separation tool of any PID is a dielectric membrane with conducting channels filled by immobiline gels of varying pH. The pH value of the surrounding aqueous solution is not equal to the pH of any of the channels. The membrane is held perpendicular to the applied electric field. Proteins are collected (trapped) in the channels whose pH values are equal to the pI of the proteins. The fast particle transport between different channels takes place due to convection in the aqueous solution. We developed a mathematical model for PID. Experiment duration is shown to be proportional to the number of different bands N (the peak capacity in standard IEF) in contrast with N(2) for usual IEF devices. This model was validated with experimental results. Parallel IEF accelerates the fractionation of proteins by their pI values (down to several minutes) allowing a more desirable collection efficiency to be achieved. The main theoretical limitation of PID resolution is the sensitivity of proteins to pH change due to the Coulomb blockade effect. The existence of a minimal pH change deltapH(min) for each type of protein is shown: deltapH(min) approximately r(-1) for globular molecules with radius r.

Parallel processing in the isoelectric focusing chip.

Investigation of isoelectric focusing (IEF) kinetics has been performed to provide the theoretical basis for miniaturization of classical IEF in immobilized pH-gradients. Standard IEF demands colinearity of the electric field and pH-gradient directions (serial devices). It is shown that the IEF separation process based on a continuous, serial pH gradient is incompatible with miniaturization of separation devices. The new realization of the IEF device by a parallel IEF chip is suggested and analyzed. The main separation tool of the device is a dielectric membrane (chip) with conducting channels that are filled by Immobiline gels of varying pH. The membrane is held perpendicular to the applied electric field and proteins are collected (trapped) in the channels whose pH are equal to the pI of the proteins. The pH value of the surrounded aqueous solution is not equal to any channel's pH. The fast particle transport between different channels takes place due to convection in the aqueous solution. The new device geometry introduces two new spatial scales to be considered: the scale of transition region from a solution to the gel in a channel and a typical channel size. The corresponding time scales defining the IEF process kinetics are analyzed and scaling laws are obtained. It is shown both theoretically and experimentally that parallel IEF accelerates the fractionation of proteins by their pI down to several minutes and enables possible efficient sample collection and purification.

Finite element simulation of Off-Gel trade mark buffering.

The protonation of an aqueous solution of two ampholytes AH and BH next to a gel buffered by immobilized acid moieties IH has been studied by finite element simulation in an iterative scheme. A ten species model has been formulated, taking into account transient diffusion and equilibrium kinetics of the two amphoteric species AH and BH, of water and of the immobilized species IH. This model has been developed to illustrate the pH evolution between an ampholyte solution and an Immobiline gel, and to study the influence of the Immobiline concentration on protons and ampholyte distributions. It has been demonstrated that a minimum initial Immobiline concentration of 10(-2) M is necessary to maintain the pH in the gel in contact with a closed chamber, when the difference between the isoelectric points of AH and BH is 4 and when the initial concentration of the ampholytes in solution is in the micromolar range. This approach provides a first theoretical framework for the recently developed Off-Gel trade mark electrophoresis.

A miniaturized multichamber solution isoelectric focusing device for separation of protein digests.

A miniaturized multichamber device was constructed for solution isoelectric focusing (IEF) separation of complex peptide mixtures. The system, based on immobilized pH gels, consisted of 96 minichambers ( approximately 75 nuL each) arranged in eight rows. Neighboring chambers in a given row were separated by short glass tubes (4 mm inner diameter, 3 mm long), within which Immobiline gels of specific pH values were polymerized. During focusing, the device was sandwiched between two supporting blocks incorporating the reservoirs for anolyte and catholyte. In principle, multiple samples could be simultaneously fractionated, each separated into 12 fractions of various pI ranges. A variety of standard peptide mixtures and tryptic digests of proteins were separated by IEF using this device, and the fractions were characterized by mass spectrometry. For a codigested nine-protein mixture, both the total number of peptides identified and the average sequence coverage were similar to the results of ion-exchange chromatography (IEC), according to matrix assisted laser/desorption/ionization--time of flight (MALDI-TOF) data. The IEF separation provided concentrated and desalted fractions, suitable for an additional separation liquid chromatography, capillary electrophoresis (LC, CE) or mass spectrometry (MS) detection without additional sample cleanup. High loading capacity was achieved for the miniaturized multichamber IEF device. Importantly, a linear correlation was found between the experimentally determined and calculated pI values of peptides.

Two-dimensional maps in soft immobilized pH gradient gels: a new approach to the proteome of the Third Millennium.

Same major improvements in proteome analysis of cytosolic and membrane proteins by two-dimensional mapping are here reported. A much improved transfer of proteins from the first to the second dimensional sodium dodecyl sulfate (SDS)-gel is obtained by simply diluting the gel matrix, normally composed of 4%T polyacrylamide in all commercially available Immobiline strips down to as low as 3%T. In the analysis of total lysates of platelets, this augmented transfer has been evaluated as being 2-3 times higher than in standard 4%T gels. A second major improvement, in the case of analysis of membrane protein preparations, has been demonstrated to consist in a delipidation step in a tertiary solvent mixture composed of tri-n-butyl phosphate:acetone:methanol in a 1:12:1 ratio. By adopting this protocol, large amounts of spectrins (240-220 kDa, filamentous proteins of the red blood cell membranes) could be transferred vs. essentially none when delipidation was omitted. The present report also confirms the importance of a reduction and alkylation step of the protein sample prior to all electrophoretic steps, including focusing in the Immobiline gel, as recently reported by Herbert et al.

Two‐Dimensional Gel Electrophoresis

While one-dimensional SDS-PAGE separates proteins on the basis of size, two-dimensional gel electrophoresis separates proteins first on the basis of isoelectric point, then on the basis of size. This method is capable of resolving 1000 to 2000 separate proteins when combined with sensitive detection methods. This unit describes methods for characterizing cell lysates by two-dimensional gel electrophoresis, including modifications for acidic and basic proteins, the use of immobilized pH gradients, and nonreducing/reducing electrophoretic separations. In addition there are support protocols for determining pH profiles of gels, casting Immobiline gels, preparing cell and tissue samples for isoelectric focusing, preparing molecular weight standards, and using two-dimensional protein databases.

Two‐Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis combines two different electrophoretic separating techniques in perpendicular directions to provide a much greater separation of complex protein mixtures than either of the individual procedures. Variations of the most common two-dimensional technique are described in this unit, namely isoelectrofocusing (IEF) and SDS-PAGE. This unit also includes support protocols describing pI standards and pH profile measurements, casting Immobiline gels, preparation of tissue culture cells and solid tissues for isoelectricfocusing, preparation of molecular weight standards for two-dimensional gels, and two-dimensional protein databases.

PH changes in Immobiline gels due to low-molecular mass ion adsorption and conditions for salt front formation during electrophoretic desorption.

A simple theoretical model of low molecular mass ion adsorption on a weak exchanger is proposed. For a system containing immobilized charges in uniform concentration the pH change values connected with neutral salt addition and followed by further washing and voltage application are evaluated. The question of stability of the moving boundary, arising in the area near the electrodes, is considered for the constant current stabilization at different values of Immobiline concentration, pK values of Immoboline and salt concentration. The threshold values of ion mobility capable of providing a sharp salt front formation are also evaluated for conditions of strong adsorption. We found that, with extremely acidic Immobilines, a sharp salt front does not arise under the latter condition, even in the case of very high Immobiline concentrations.

Isoelectrophoretic Characterization of Pseudomonas Cytochrome Oxidase/Nitrite Reductase and Its Heme d 1-Containing Domain

The cytochrome oxidase/nitrite reductase of Pseudomonas aeruginosa has been purified to homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When this "homogeneous" protein is subjected to electrophoretic titration curve analysis in ampholines or to isoelectric focusing in immobilized pH gradient gels it is resolved into several bands, each of which possesses the olive-green color of the holoenzyme. Although the patterns of resolution replicate for a given enzyme preparation differences occur among different preparations. Furthermore, storage for several months at −20°C leads to an increase in the number of isoelectrophoretic forms. All preparations, however, have two primary bands, one with a p I of 6.97 and the other of 7.02. Both these bands possess significant cytochrome oxidase activity after elution from the gels. When each of the primary bands is eluted and again subjected to iso-electric focusing under the same conditions as before, each band interconverts into two bands with p Is of 6.97 and 7.02. The addition of the ligand cyanide to the holoenzyme produces a shift in the p I of the two bands to p Is 7.04 and 7.12 while the addition of nitrite shifts some of the band at p I 6.97 into that at p I 7.02. The heme d 1 -containing dipeptide of the enzyme, produced by treatment with subtilisin, also exhibits considerable heterogeneity upon electrophoretic titration curve analysis and by isoelectric focusing in immobiline gels. Possible explanations for the observed isoelectrophoretic behavior in terms of protein conformation and heme chemistry are discussed.

Characterization of Immobiline membranes for application in a multicompartment electrolyzer for protein purification

The efficient use of preparative protein purification in a multicompartment electrolyzer with Immobiline membranes depends on the knowledge of membrane characteristics. For that purpose, an experimental investigation of the effects of ionic charges on the membrane characteristics has been carried out through the measurements of membrane swelling and conductance. We also investigated the effects on the electrolyzer behaviour of operating parameters such as the Immobiline concentration and the presence of ion-exchange membranes. Data show that polyacrylamide gel degree of swelling is strongly dependent upon the pH and the ionic strength of the bathing solution as well as on the type and molarity of charges incorporated in the gel. The conductance of supported Immobiline gels in contact with uni-univalent chloride solutions has been measured by means of a mercury cell. The membrane conductance is also influenced by the ionic strength of the equilibrium solution and the presence of weak ionizable groups in the gel matrix. This study has demonstrated the close link between electrochemical and electromechanical properties of Immoboline membranes.

Characterization of a new human apolipoprotein A-I Yame by direct sequencing of polymerase chain reaction-amplified DNA.

A new genetic variant apolipoprotein (apo), A-I Yame, was discovered during screening for apoA-I genetic variants with isoelectric focusing gel electrophoresis. To investigate the structural abnormality of apoA-I Yame, we amplified the apoA-I gene isolated from the proband's peripheral blood leukocytes with the polymerase chain reaction (PCR) method and directly sequenced these PCR fragments. ApoA-I Yame was found to have aspartic acid (GAC) substituted by tyrosine (TAC) at residue 13. We also identified this substitution by an automated DNA sequencer. This substitution was confirmed with amino acid sequencing of the isolated apoA-I Yame by Immobiline gel electrophoresis. This combined method, direct PCR from genomic DNA-derived individual peripheral blood leukocytes and subsequent direct sequencing, can be used to identify the entire sequence of apoA-I in a short period of time. Furthermore, with this method, it is possible to identify both alleles in heterozygous individuals.

Preparative isoelectric focusing in ampholine electrofocusing columns versus immobiline polyacrylamide gel for the purification of biologically active leukoregulin

Preparative vertical and rotating horizontal (Rotofor) ampholine column and immobiline flat bed polyacrylamide gel isoelectric focusing were evaluated for the isolation of the biologically active acidic form of leukoregulin, a 50,000-Da glycoprotein lymphokine with tumor growth inhibitory activity. Leukoregulin secreted by normal human lymphocytes was concentrated by 10,000 nominal molecular weight size exclusion ultrafiltration and by DEAE anion exchange chromatography using step elution with 0.02 m Tris-HCl: 0.1 m NaCl, pH 7.4. Preparative isoelectric focusing was carried out in a 110-ml vertical column containing 1% ampholines in a pH 4–6 gradient at 15 W constant power for 16–18 h, in a Rotofor 55-ml horizontal column containing 2% ampholines in a pH 4–6 gradient at 12W constant power for 4–6 h, or in an immobiline pH 4.5–6.5 gradient within a 5% polyacrylamide 120 × 110 × 5-mm flat bed gel at 3 W constant power for 16–18 h. Recovery of biologically active leukoregulin from the vertical and horizontal ampholine columns was similar. The pH 4.9–5.2 fractions from the Rotofor ampholine column contained 4–7% and the fractions from the immobiline gel contained 4% of the leukoregulin activity applied to the electrofocusing column or gel, respectively. Analytical immobiline isoelectric focusing of the leukoregulin in the pH 4.9–5.2 fractions from the Rotofor column demonstrated that a single silver staining band with a p I of 5.1 can be obtained by this rapid method of preparative isoelectric focusing.

Use of polybuffer as carrier ampholytes in mixed-bed immobiline gels for isoelectric focusing

In mixed-bed, carrier ampholyte-Immobiline gels, a primary, insolubilized pH gradient is admixed with a secondary, soluble pH gradient generated by amphoteric buffers. The latter are the standard carrier ampholytes (e.g. Ampholine, Pharmalyte, Biolyte, Servalyte), used in conventional isoelectric focusing, admixed to Immobiline gels in levels of ∼0.5–1%. It is here shown that polybuffers 96 (covering the pH 6–9 range) and 74 (covering the pH 4–7 interval) used as eluents in chromatofocusing, can effectively substitute the standard carrier ampholytes with considerable savings (they are 1 16 th as expensive as the latter chemicals).

The influence of the support material used on band sharpness in Immobiline gels

To achieve excellent isoelectrofocusing results with the use of Immobiline gels, the quality of the support is essential. Effects such as diffuse bands, missing protein fractions, streaking and trailing could originate from a support of inadequate quality. Two different treatments to eliminate these effects were evaluated. A washing procedure improves the band sharpness, and addition of carrier ampholytes eliminates streaking and trailing.

Protein desalting by isoelectric focusing in a segmented immobilized pH gradient

We have recently described an apparatus for protein purification based on a segmented Immobiline gel, having one or more liquid interlayers in between. The principle is entirely new, as it is based on keeping the protein of interest isoelectric, in a flow chamber, and focusing the impurities in an Immobiline gel. For this, a hydraulic flow is coupled orthogonally to an electric flow, sweeping away the non-isoelectric impurities from the recycling chamebr. We now demonstrate that the present apparatus can be efficiently used for protein desalting. Hemoglobin A samples, containing 50 mM NaCal or 50 mM ammonium acetate, could be efficiently desalted in 2 h of recycling, after which the total salt content had decreased to less than 0.005 mM (a salt decrement of more than 10 000 fold the initial input). However, with polyprotic buffers (sulphate, citrate, phosphate, oligoamines) the desalting process was much slower, typically of the order of 20 h, possibly due to interaction of these species with the surrounding Immobiline matrix. In this last case, outside pH control (e.g. with a pH-stat) is necessary during protein purification, as due to the faster removal of the monovalent counterion, the solution in the recycling chamber can become rather acidic or alkaline. It is demonstrated that the 2 extremities of the Immoboline segments facing the sample recycling chamber act indeed as isoelecctric membranes, having a good buffering capacity, preventing the protein macroion from leaving the chamber of continously titrating it to its isoelectric point.