Moving and stationary boundaries in immobilized pH gradients

The relations describing the concentration changes at moving boundaries in a medium containing bound, buffering group are derived for a system which, except for hydrogen and hydroxyl ions, contains one anionic and one cationic mobile constituent. The relations found have been used to calculate concentrations and conductivities in zones developing in immobilized pH gradients. Assumptions used in the calculations as well as conductivity ratios between zones have been experimentally controlled and were found to reasonably agree with expectations. It is also shown how difference in transference numbers between sample droplet and gel will cause concentration and pH changes at the gel-sample droplet interfaces and it is explained how these changes are related to ionic concentrations in the gel. The high concentration zone generated at one of the interfaces will be transported into the gel. This transport has been numerically simulated and experimentally verified. The low concentration generated at the opposite interface will cause titration impeding sample entrance in the gel through this interface even when the gel contains ions other than H+ or OH- transported towards the interface. The described phenomena explain the dependence of lateral spreading, precipitation at the application site as well as streaking and smearing along sample lanes, on the type and concentration of low molecular weight ions originally present in the gel.     

Elimination of sample diffusion and lateral band spreading in isoelectric focusing employing ready-made immobilized pH gradient gels.

A simple procedure for the elimination of lateral sample as well as band spreading in precast, ready-made immobilized pH gradient gels is described. Round or rectangular holes are punched in the dry polyacrylamide gels prior to rehydration. The generated wells proved suitable for application of samples containing surfactants such as Nonidet P-40 or 3-[(3-cholamidopropyl) dimethylammonio]-1- propane-sulfonate (CHAPS). Lateral band spreading and precipitation of samples containing up to 9.5 M urea could be completely eliminated by this method.     

Two-dimensional gel electrophoresis of platelet polypeptides with immobilized pH gradients in capillary tubes

Two-dimensional electrophoresis (2-DE) with immobilized pH gradient (IPG) gels in capillary tubes was used in the first-dimensional isoelectric focusing (IEF) for the separation of human platelet polypeptides. Two types of IPG tube gels, pH ranges 4-8 and 7-10, containing 8 M urea, 1% Nonidet P-40 and 0.1% pH 3.5-10 Ampholine carrier ampholytes (CA) were prepared by a simple method not requiring special equipment. The addition of CA to both gel and sample solutions was essential in the tube gel IPG system. Proteins were visualized by a modification of Wray's silver-staining technique. The degree of resolution and the number of spots observed on an IPG 2-DE gel with pH 4-8 were comparable with those obtained with O'Farrell's high-resolution 2-DE. Approximately 200 basic polypeptides, which are difficult to separate by conventional CA-based IEF 2-DE or the non-equilibrium pH gradient system, were well resolved by 2-DE with a pH 7-10 IPG tube gel in the first-dimension. The gel patterns with either pH gradient 4-8 or 7-10 were highly reproducible among gels prepared and run simultaneously. These results demonstrated the potential and usefulness of the 2-DE system with IPG gels in capillary tubes.     

Approach to stationary two-dimensional pattern: influence of focusing time and immobiline/carrier ampholytes concentrations

Horizontal two-dimensional (2-D) electrophoresis with immobilized pH gradients (IPG) in the first dimension for buffer soluble proteins and for complex proteins solubilized in the presence of Nonidet P-40 (G?rg et al., Electrophoresis 1987, 8, 45-51), has been extended to analyze basic proteins of yeast cells focused under non-equilibrium and equilibrium conditions. Transient state isoelectric focusing (IEF) in IPG gels revealed sample smearing and background staining, displaying horizontal streaks in the resultant 2-D patterns. Inclusion of 0.5% carrier ampholytes (CA) to the IPG gel (IPG-CA), resulted in the formation of many sharp protein bands after transient state IEF with resultant distinct spots in the 2-D patterns; however, resolution was poor and the gel contained heavy background staining. With prolonged focusing time, background staining disappeared and there was less difference in the final steady state IEF patterns obtained with IPG and IPG-CA. Reduction of the Immobiline concentration to one third the manufacturer's recommended amount did not improve IEF resolution with respect to streaking and background staining under either transient state or equilibrium conditions. In general, spot intensities were less on 2-D gels using diluted IPG gels than with "standard" IPG gels. Optimization of 2-D electrophoresis with IPGs in the first dimension was strongly related to IEF conditions. The use of IPG gels focused to equilibrium should not only improve inter-gel reproducibility and resolution but also the quality of the final 2-D patterns with respect to background staining and horizontal streaking.     

Two-dimensional gel electrophoresis using immobilized pH gradient tube gels

An apparatus for the preparation of gels for immobilized pH gradient isoelectric focusing (IPG) in glass tubes was developed. Using this apparatus, the highly reproducible immobilized pH gradient can be formed with Immobilines in polyacrylamide gels, and IPG gels at all possible pH ranges can be easily prepared at low cost. The IPG tube gels in the first dimension in two-dimensional gel electrophoresis was used to separate and identify a number of rice embryo proteins in the proteome analysis. There was no difference in resolution of proteins between the tube gels and the commercially available slab gels; after electrophoresis, however, we could efficiently obtain a larger amount of the purified proteins from the tube gels than from the slab gels.     

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis without a stacking gel: use for high sample throughput

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis without a stacking gel minimizes lateral spreading of protein when samples are applied in agarose wells and allows high sample throughput (6 samples/cm gel width). The method is simple and convenient to use and gives comparable resolution to the standard method with 4-20% or 6-30% polyacrylamide gradient gels. Best results are obtained when the upper zone of the separating gel is of low polyacrylamide concentration. This indicates a need for the molten agarose to penetrate and anneal with the separating gel.     

Immobilized pH gradients: effect of salts, added carrier ampholytes and voltage gradients on protein patterns

Salts formed from strong acids and bases (e.g. NaCl, Na2SO4, Na2HPO4), present in a protein sample applied to an immobilized pH gradient (IPG) gel, induce protein modification (oxidation of iron moiety in hemoglobin) already at low levels (5 mM) and irreversible denaturation (precipitation) at higher levels (greater than 50 mM). This effect is due to production of strongly alkaline cationic and strongly acidic anionic boundaries formed by the splitting of the salt's ion constituents, as the protein zone is not and can not be buffered by the surrounding gel until it physically migrates into the gel matrix. Substitution of "strong" salts in the sample zone with salts formed by weak acids and bases, e.g.. Tris-acetate, Tris-glycinate, Good's buffers such as (N-[2-acetamido]-2-iminodiacetic acid (ADA), (2-[(2-amino-2-oxoethyl)-amino] ethanesulfonic acid (ACES), (3-[N-morpholino]propane sulfonic acid (MOPS), essentially abolishes both phenomena, oxidation and irreversible denaturation. Suppression of "strong" salt's effects is also achieved by adding, to the sample zone, carrier ampholytes in amounts proportional to the salt present (e.g. by maintaining a salt: carrier ampholytes molar ratio of at least 1:1). This suppression is due to the strong buffering power of the added carrier ampholytes, able to counteract drastic pH changes in the two moving boundaries. A reduction of these deleterious effects of strong salts is also achieved when the IPG run is performed at low voltage for a prolonged time (4 h at 500 V instead of only 1 h at 500 V, before switching to high-voltage settings). Guidelines are given for trouble-free IPG operations.     

A simple method to remove contaminating salt from IPG strips prior to IEF

In this study, Saccharomyces cerevisiae yeast lysate is used to demonstrate how a simple wash procedure can improve IEF of IPG strips passively rehydrated in the presence of NaCl. By performing three 10 min washes after IPG strip rehydration and before IEF, corresponding second-dimensional gels from strips containing NaCl look similar to control strips while the second-dimensional gels of unwashed strips contains streaks and spaces devoid of protein. Up to 500 mM NaCl was added to the yeast lysate and successfully focused following this wash regime. Protein loss due to the washes was determined to be minimal by comparing replicates of washed and unwashed strips and analyzing the densities of their corresponding second-dimensional gel spots. In the event of unknown salt contamination, indicated by low voltage during focusing, it is possible to stop focusing, wash the strips, and then continue focusing with acceptable second dimension results.     

Modified silver staining for immobilized pH gradients.

Silver development of gels containing an immobilized pH gradient has proved difficult so far because the bonded buffers (especially the tertiary amino acrylamido derivatives) tend to absorb silver ions with a resultant heavy background of increasing darkness from the anode to the cathode. We report a variant of silver staining in which thiosulfate is used twice: (i) prior to silver impregnation, at the millimolar level, to enhance sensitivity, and (ii) during development, at the micromolar level, to decrease the background.     

Trypsin activity assay in substrate-specific one- and two-dimensional gels: a powerful method to separate and characterize novel proteases in active form in biological samples

To separate and identify the proteases, a substrate-specific, sensitive assay in sodium dodecyl sulfate (SDS)-polyacrylamide gels after two-dimensional (2-D) electrophoresis has been developed. This method allows simultaneous determination of protease cleavage specificity, molecular weight, isoelectric point, and if necessary, amino acid sequencing. After isoelectric focusing in immobilized pH gradient (IPG) strips (pH 6-11) (first dimension), trypsin was electrophoresed in 12% SDS polyacrylamide gels (second dimension) copolymerized with Boc-Gln-Ala-Arg-MCA (4-methyl-coumaryl-7-amide). The gels were washed in cold 2.5% Triton X-100 and water, and incubated in assay buffer (6.3 mM Bicine, 100 mM NaCl). Trypsin cleavage of the peptide-MCA generated fluorescent 7-amino-4-methyl-coumarin. In 1-D gels, as low as 500 pg trypsin could be detected and trypsin band volumes correlated linearly with the amounts of trypsin (R(2) = 0.999). In 2-D gels, the lowest amount of trypsin detected was 1 ng. The linear regression of spot volume and loading amount was still good (R(2) = 0.974). To optimize renaturation conditions, 5x5 min washes with 2.5% Triton X-100 and water, respectively, gave the strongest band volume. For fluorescence development, an assay buffer at pH 9 was the best; incubation at 37 degrees C for 30 min was sufficient. The method has application for identifying novel proteases as it does not rely on antibodies.     

Isoenzyme analysis of lichen algae in immobilized pH gradients

A base for a modern species' concept of chlorococcal algae can be obtained not by morphological analysis, but by biochemical characters, e.g. isoenzyme banding patterns. From isolated lichen algae of the genus Trebouxia de Puymaly a set of five such enzymes has been studied by isoelectric focusing in immobilized pH gradients (IPG): phosphoglucomutase, phosphoglucose isomerase, malate dehydrogenase, mannitol dehydrogenase and leucine aminopeptidase. The first four are resolved into isoforms in a pH 4-7 IPG interval, while the last one is analyzed in an IPG pH 3.5-5 span. The patterns are specific for distinct populations, inter- and intraspecifically varying in dependence from their geographical distribution or the lichen species from which they have been isolated. Their limited heterogeneity (one to four isoforms) suggests that they are the products of specific genes rather than artefacts of the extraction procedure or the IPG analysis. Sharp isozyme patterns can only be obtained in a mixed-bed, carrier-ampholyte (CA)-IPG gel and by anodic application, suggesting that the recently proposed mechanism of hydrophobic protein-IPG matrix interaction (Electrophoresis, 1987, 8, 62-70) is fully operative here. As an additional mechanism, it is proposed that, in some cases, CA might simply act, when added to an IPG gel, by buffering, in the transient state, the sample zone before the protein migrates from the liquid phase into the IPG matrix.     

Isoelectric focusing in long immobilized pH gradient gels to improve protein separation in proteomic analysis.

The number of protein spots detected on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) gels increases as the gel size increases. The largest commercially available systems resolve a few thousand spots, being only a fraction of the total proteome. We have developed an extremely long isoelectric focusing (IEF) system aimed at more complete protein profiling. The system is especially well suited to sensitive detection methods, such as radioactive detection. The major constraint preventing progress in this area has been the inability to create an even density gradient during the immobilized pH gradient (IPG) casting process. We demonstrate for the first time that this constraint can be effectively overcome, to enable greatly increased IEF separating power with all the advantages of IPG technology,     

Phosphoprotein electrophoresis in the presence of Fe(III) ions

Preparation of affinity polyacrylamide gels containing immobilized Fe(III) ions for the separation of proteins exhibiting metal ion binding properties is described. The presented method enables uniform distribution of immobilized metal ions in the affinity part of the polyacrylamide separating gel. Affinity gels prepared by this way are suitable to follow the effect of different concentrations of metal ions immobilized in polyacrylamide gel on a protein electrophoretic behavior. Polyacrylamide gels containing immobilized Fe(III) ions were used to study the electrophoretic behavior of two model proteins differing in their phosphate group content: chicken ovalbumin and bovine α‐casein. For the electrophoretic separation, both the native and the denaturating conditions were used.     

An optimized procedure for detection of proteins on carrier ampholyte isoelectric focusing and immobilized pH gradient gels with imidazole and zinc salts: its application to the identification of isoelectric focusing separated isoforms by in-gel proteolysis and mass spectrometry analysis.

A method for the characterization of proteins separated by isoelectric focusing in carrier ampholytes (CA-IEF) or immobilized pH gradient (IPG) gels by in-gel digestion and mass spectrometry is described. Proteins are detected by an improved imidazole-Sodium dodecyl sulfate (SDS)-zinc staining adapted for IEF and IPG gels. Sensitivity is close to that of mass spectrometry-compatible silver staining, but simpler and faster. Proteins were digested in imidazole-SDS-zinc stained CA-IEF and IPG gels in the presence of a zinc-chelating agent. Mass spectra were clearly interpretable as carrier ampholytes which were efficiently removed before digestion; high-sequence coverage that allowed isoform characterization was obtained by analyzing both the aqueous and the organic phase extracts.     

Top-down, bottom-up, and side-to-side proteomics with virtual 2-D gels

Intact protein masses can be measured directly from immobilized pH gradient (IPG) isoelectric focusing (IEF) gels loaded with mammalian and prokaryotic samples, as demonstrated here with murine macrophage and Methanosarcina acetivorans cell lysates. Mass accuracy and resolution is improved by employing instruments which decouple the desorption event from mass measurement; e.g., quadrupole time-of-flight instruments. MALDI in-source dissociation (ISD) is discussed as a means to pursue top-down sequencing for protein identification. Methods have been developed to enzymatically digest all proteins in an IEF gel simultaneously, leaving the polyacrylamide gel attached to its polyester support. By retaining all gel pieces and their placement relative to one another, sample handling and tracking are minimized, and comparison to 2-D gel images is facilitated. MALDI-MS and MS/MS can then be performed directly from dried, matrix-treated IPG strips following whole-gel trypsin digestion, bottom-up methodology. Side-to-side proteomics, highlighting the link between virtual and classical 2-D gel electrophoresis, is introduced to describe a method whereby intact masses are measured from one side (the IEF gel), while proteins are identified based on analyses performed from the other side (the SDS-PAGE gel).     

Direct observation by laser scanning confocal microscopy of microstructure and phase migration of PVC gels in an applied electric field

The fluorescent probe lucigenin was incorporated in poly(vinyl chloride) (PVC) gels, and laser scanning confocal microscopy (LSCM) was used to clarify the internal structures of the gels. From the two-dimensional and three-dimensional information by LSCM, we first observed the internal structure of the PVC gel at a wet status, where the PVC gels comprised a polymer-rich phase and a polymer-poor phase uniformly with a three-dimensional network structure. After an electric field was applied, an effect of the electric field resulted in the change of internal structure in the gels. The polymer-poor phase moved from the cathode to the anode and the polymer-rich phase formed linelike arrangement between electrodes due to the attraction force. On the other hand, the freeze-dried PVC gels with/without in-situ dc voltage casting were particularly fabricated to confirm above results by the field emission scanning electron microscopy (FE-SEM). It was found that many craters remained on the surface of the gel near the anode due to sublimation in freeze-drying. This phenomenon did not appear on the surface near the cathode. The results of in-situ dc voltage casting also suggested that a substantial amount of polymer-poor phase was moved and fixed at the anode. Thus, results of both LSCM and in-situ dc voltage casting corresponded to the effect of electric field on PVC gels and provided a convincing evidence for the interpretation of the deformation mechanism of PVC gel actuators by an applied electric field.     

Diffusion coefficients of proteins in carrier ampholyte versus immobiline gels

The apparent diffusion coefficients of proteins in carrier ampholyte isoelectric focusing (CA-IEF) and in immobilized pH gradients (IPGs) are strongly dependent on the amount of buffering ions present in the system. However, whereas in CA-IEF increased levels of ampholytes facilitate diffusion, in IPGs they strongly quench it. It is concluded that a protein in an IPG matrix is isoelectric but not isoionic, in the sense that it forms a salt with the surrounding ions bound to the polyacrylamide matrix. This salt formation is beneficial as it greatly increases protein solubility at the pI. It is suggested that, when performing zymograms in situ, the IPG gel should contain at least twice the standard amount of Immobiline, so as to keep sharp enzyme bands even with prolonged incubation periods.     

An optimized procedure for solubilization, reduction, and transfer of human breast cancer membrane-enriched fraction by 2-DE

The separation of integral and peripheral membrane proteins is still a challenge, although many achievements have been made in the 2-DE-based membrane proteomics. Using a human breast cancer cell line, MCF-7, we investigated the influences of Tris, reducing reagents, cup loading, and SDS on membrane protein solubilization and separation by 2-DE. The addition of Tris to the sample solution improved the solubilization of the membrane-enriched fraction, and the best-quality gel patterns were obtained at 20 mM Tris. Tributylphosphine (TBP), a reducing agent, was not optimum in the 2-DE process because it not only decreased the solubilization of hydrophobic proteins but also caused some proteins, such as hsp60, prohibitin, and actin, to be resolved to a string of spots. However, when combined with DTT, TBP could improve the resolution of 2-DE patterns. Cup loading significantly facilitated the entrance of membrane proteins into IPG strips and over 1000 protein spots with high resolution were visualized. Adopting this strategy, an ATP synthase alpha chain was resolved into two adjacent spots for the first time in 2-DE gel patterns through the adding DTT in the middle of the IEF. A high SDS concentration in the equilibration buffer enhanced the transfer and increased the staining intensity of 50% of the protein spots in the gels, but also resulted in losses of some spots.     

Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels

Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5% native polyacrylamide gel electrophoresis (PAGE) gels or 15% sodium dodecyl sulfate (SDS)-PAGE gels with or without further purification by a 2',5'-ADP Sepharose 4B affinity column. For SDS-PAGE gels, the SDS was removed first by washing twice with 25% isopropanol in 10 mM Tris-HCl (pH 7.9) for 10 min. The gel was then dipped in a 50 mM Tris-HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min. The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5-10 min. The contrast between the clear zone of GR activity and the purple background was found in both native and SDS-PAGE gels. This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources. Under reduced SDS-PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast. This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.     

A comparison of three commercially available isoelectric focusing units for proteome analysis: the multiphor, the IPGphor and the protean IEF cell

We tested and compared three different commercially available instruments for isoelectric focusing for proteome analysis by two-dimensional protein electrophoresis. These instruments, the Multiphor, the IPGphor, and the Protean IEF cell, were used with 18 cm immobilized pH gradient strips and run under various conditions. The total number of spots and features was obtained by Melanie software (Bio-Rad Laboratories) and separately by visual inspection. The Multiphor consistently resulted in the highest number of spots detected per gel independent of sample type, immobilized pH gradient (IPG) and method to calculate the number of spots. The Protean IEF cell had the next highest number of spots detected per gel. In the experiments performed, the IPGphor afforded good reproducibility in the total number of Melanie-detected spots from gel to gel while the Protean IEF cell offered better reproducibility in the total number of manually detected spots from gel to gel. Among gels run with the different instruments, differences in the quality of the ammoniacal silver stain were also observed. A measure of quantitative reproducibility suggests that the Protean IEF cell, which was the easiest instrument to use, performs better than the other instruments, although all three instruments had demonstrated good quantitative reproducibility in the experiments performed.