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.     

Mass spectrometric imaging of immobilized pH gradient gels and creation of "virtual" two-dimensional gels

We have developed a matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) based technique for the detection of intact proteins directly from immobilized pH gradient gels (IPGs). The use of this technique to visualize proteins from IPGs was explored in this study. Whole cell Escherichia coli extracts of various loadings were separated on IPGs. These IPGs were processed to remove contaminants and to achieve matrix/analyte cocrystallization on the surface of the gel. Mass spectra were acquired by scanning the surface of the gel and were assimilated into a "virtual" two dimensional (2-D) gel. This virtual 2-D gel is analogous to a "classical" 2-D gel, except that the molecular weight information is acquired by mass spectrometry rather than by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This mass spectrometry (MS) based technology exemplifies a number of desirable characteristics, some of which are not attainable with classical two-dimensional electrophoresis (2-DE). These include high sensitivity, high reproducibility, and an inherently higher resolution and mass accuracy than 2-D gels. Furthermore, there is a difference in selectivity exhibited between virtual 2-D gels and classical 2-D gels, as a number of proteins are visible in the virtual gel image that are not present in the stained gels and vice versa. In this report, virtual 2-D gels will be compared to classical 2-D gels to illustrate these features.     

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)     

Pouring wide-range immobilized pH gradient gels with a window of extremely flattened slope

A gradient mixing device has been designed to pour polyacrylamide gels with a wide-range immobilized pH gradient including a "window" of extremely flattened slope. The device consists of an IBM-compatible personal computer controlling 8 step-motor-driven burettes, with four producing a density gradient from glycerol and delivering acrylamides and catalysts for gel polymerization, two delivering Immobilines for a wide-range pH gradient and the other two burettes responsible for the delivery of Immobilines for a partial pH range inside the wide range. The effect on a complex separation pattern of proteins with a wide range of pI is that resolution can be increased reproducibly to any reasonable extent at any location of the separation pattern.     

Enantiomer resolution in immobilized pH gradient gels via inclusion of a chiral discriminator

The separation of enantiomeric forms of dansylated amino acids by isoelectric focusing in immobilized pH gradients (IPG) is demonstrated for the first time. Separations occur in a pH 3.0-4.0 IPG interval, in presence of 7Murea, 10% methanol and 60 mM beta-cyclodextrin (CD) as chiral discriminator. It is found that the inclusion complex formed between the D-form and CD has a lower pI than the uncomplexed form (delta pI = 0.05 for DL-Phe and delta pI = 0.025 for DL-Trp); from this, it is calculated that the pK of the tertiary amino group in the dansyl moiety is lowered by 0.1 pH unit in the former case (D-Phe) and by 0.05 in the case of D-Trp (both values referring to 60 mM CD gels). For some racemates (e.g., DL-Phe) the separation mechanism is still operative with CD concentrations as low as 20 mM. In our system 60 mM CD appears to be the solubility limit of CD. As the complex is stable in the electric field for at least 15 h, this separation mechanism could be exploited for purifying large quantities of pure D and L forms from racemates in multicompartment electrolyzers with isoelectric Immobiline membranes.     

Proteome analysis using isoelectric focusing in immobilized pH gradient gels followed by mass spectrometry

Over the past several years, a large effort has been focused on improvements of two-dimensional (2-D) gel electrophoresis-based proteomics technology, and on development of novel approaches for proteome analysis. Here, we describe the application of an alternative strategy for the analysis of complex proteomes. The strategy combines isoelectric focusing in immobilized pH gradient strips (in-gel IEF), mass spectrometry (MS), and bioinformatics. A protein mixture is separated by in-gel IEF, and the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the tryptic peptides are subjected to liquid chromatography-nanoelectrospray-quadrupole ion-trap tandem mass spectrometry (LC-ESI-MS/MS). The LC-ESI-MS/MS data are used to identify the proteins through searches of a protein sequence database. Using this in-gel IEF-LC-MS/MS strategy, we have identified 127 proteins from a human pituitary. This study demonstrates the potential of the in-gel IEF-LC-MS/MS approach for analyses of complex mammalian proteomes.     

Revisiting electroblotting of immobilized pH gradient gels: a new protocol for studying post-translational modification of proteins

Currently, one of the most important techniques in proteome analysis is two-dimensional electrophoresis that is widely used for separation of thousands of different protein spots. Nevertheless, characterization of special aspects in protein patterns, e.g., separation of protein isoforms generated by post-translational modifications, requires individual detection methods, e.g., immunoblotting. Blotting of proteins after fractionation in immobilized pH gradients has always caused some problems. In this paper we present an optimized protocol for immunoblotting after isoelectric focusing using immobilized pH gradient (IPG) strips cast on Net-Fix as an internal support that is permeable to electric current. The focusing procedure can be carried out in commonly used IPG systems, e.g., the IPGphor by Amersham Biosciences, where electrically assisted rehydration can be performed. This may be of interest for many laboratories, because the same system as used for the first dimension of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is involved. As an example, we describe separation and detection of up to seven isoforms of recombinant erythropoietin beta using semidry blotting of IPG strips and visualization by chemiluminescence detection.     

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

We previously demonstrated the separation of proteins by isoelectric focusing (IEF) over pH 4-8 immobilized pH gradients (IPGs) over 54 cm (Poland et al., Electrophoresis 2003, 24, 1271). Here we show that similar results can be conveniently achieved using commercially available IPGs of appropriate pH ranges positioned end-on-end in series during electrophoresis, which we term "daisy chain IEF". Proteins efficiently electrophorese from one IPG to another during IEF by traversing buffer-filled porous bridges between the serial IPGs. A variety of materials can function as bridges, including paper, polyacrylamide gels or even IPGs. The quality of two-dimensional (2-D) protein patterns is not apparently worse than that generated by conventional IEF using the same individual IPGs. A major advantage of this method is that sample is consumed efficiently, without the requirement for preliminary steps, such as chamber IEF. This advantage is pronounced when working with extremely limited sources of samples, such as with clinical biopsies or cellular subfractions. The present study was limited by the commercial availability of suitable pH gradients. Proteomics analyses could be further improved if commercial vendors would manufacture IPGs with suitable pH ranges to achieve high resolution (approximately 100 cm) IEF separation of proteins in one electrophoretic step over the pH range 2-12.     

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,     

Isoelectric focusing of group specific component in semen and vaginal stains on ultrathin immobilized pH gradient gels

Subtyping of the group specific component in secretions of semen and vaginal fluids is impossible with conventional detection systems. By means of a highly sensitive alkaline-phosphatase secondary antibody system the group specific component can reliably be detected in semen stains. Results with vaginal swabs were inconsistent and in saliva stains Gc activity could not be detected.     

Proteomic profiling combining solution-phase isoelectric fractionation with two-dimensional gel electrophoresis using narrow-pH-range immobilized pH gradient gels with slightly overlapping pH ranges

This paper describes a simple new approach toward improving resolution of two-dimensional (2-D) protein gels used to explore the mammalian proteome. The method employs sample prefractionation using solution-phase isoelectric focusing (IEF) to split the mammalian proteome into well-resolved pools. As crude samples are thus prefractionated by pI range, very-narrow-pH-range 2-D gels can be subsequently employed for protein separation. Using custom pH partition membranes and commercially available immobilized pH gradient (IPG) strips, we maximized the total separation distance and throughput of seven samples obtained by prefractionation. Both protein loading capacity and separation quality were higher than the values obtained by separation of fractionated samples on narrow-pH-range 2-D gels; the total effective IEF separation distance was ~82 cm over the pH range pH 3–10. This improved method for analyzing prefractionated samples on narrow-pH-range 2-D gels allows high protein resolution without the use of large gels, resulting in decreased costs and run times.      

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.     

Macroporous polyacrylamide-based monolithic column with immobilized pH gradient for protein analysis

Monolithic materials were prepared in capillaries by in situ polymerization of acrylamide, glycidyl methacrylate, and N,N'-methylenebisacrylamide in the presence of 1,4-butanediol, dodecanol, and DMSO as porogens. With Ampholine attached to the surface of the porous monolith via epoxide groups, a monolithic-IPG (M-IPG) was formed and showed good mechanical and chemical stability. With such a column immobilized by Ampholine 3.5-10, IEF-MIX 3.6-9.3 was separated and good linearity was obtained. The CIEF behavior of M-IPG was evinced by comparing the current with that in the open tubular capillary. In addition, the protein mixtures excreted from lung cancer cells of rats were analyzed with such a new M-IPG column.     

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.     

新型固定化pH梯度毛细管等电聚焦方法用于蛋白分离

通过化学键合建立一种固定化pH梯度的方法,用于毛细管等电聚焦分离蛋白质.采用微流控泵驱动毛细管内的聚焦区带,通过调节泵的流量,从而调节聚焦区带的迁移速度.该方法避免了自由溶液聚焦时两性电解质所带来的影响,实现了高灵敏度及检测波长自由选择等优点,适用于两步法毛细管电泳等电聚焦分离蛋白质等两性电解质.本文考察了对牛血清白蛋白和血红蛋白两种蛋白质混合物的分离,证明了该方法可行.     

Repeatedly usable immobilized pH gradient in a monolithic capillary column

Glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) were used to synthesize a monolithic capillary column containing reactive epoxy groups. Glutaraldehyde was introduced and linked to the monolith after a process of amination. An aqueous solution of commercial carrier ampholytes (CAs, Ampholine) was focused in such a polymer column. The primary amino groups of CAs reacted with glutaraldehyde along the capillary. CAs were immobilized at different positions in the column according to their isoelectric points (pI), resulting in a monolithic immobilized pH gradient (M-IPG). Isoelectric focusing (IEF) was performed without CAs in such an M-IPG column. Due to the covalent attachment of the CAs this M-IPG can be repeatedly used after its preparation. Good stability, linearity, and reproducibility were obtained.     

基于固定化pH梯度整体材料的芯片自由流等电聚焦电泳模式的构建

芯片自由流电泳对于来源稀少的重要生物样品的连续预分级和微制备具有重要的意义。本文在自由流芯片的微分离腔内,通过原位光引发聚合反应制备了聚丙烯酰胺整体材料,并进行了pH梯度的固定化,从而构建了基于固定化pH梯度整体(M-IPG)材料的芯片自由流等电聚焦模式(μFF-IEF)。利用该新型分离模式,实现了异硫氰酸荧光素(FITC)标记的最小等电点相差0.33的甘氨酸、脯氨酸和赖氨酸混合物的分离,且分离结果优于传统的μFF-IEF。实验结果表明,通过发展基于M-IPG材料的μFF-IEF模式,不仅可以避免在缓冲溶液中添加两性电解质对后续采用其他模式分离和质谱鉴定的干扰,而且可以获得较高的分离和富集能力,有望在微量样品的连续分离和制备方面发挥重要作用。     

Preparation of polyacrylamide based monolith with immobilized pH gradient and its application for protein analysis

Monolithic materials were prepared in capillaries by in situ polymerization of acrylamide, glycidyl methacrylate and N,N′-memylenebisacrylamid in the presence of trinary porogens, including 1,4-butanediol, dodecanol and dimethyl sulphoxide. With Ampholine immobilized on the monolith by chemical bonding according to their pIs, the monolithic immobilized pH gradient (M-IPG) was prepared, and applied to the separation of four standard proteins. Compared with polyacrylate based M-IPG, the hydrophilicity of the new material was improved. It could not only avoid the adsorption of proteins, but also make the synthesized procedure simple, which showed great potential in the analysis of proteins.     

Formulations for immobilized pH gradients including pH extremes

Formulations are given both for narrow (less than 2 pH units) and for wide range (up to 8 pH units) immobilized pH gradients, spanning between pH 2.5 and pH 11. The contribution from water to the buffering power (beta) at these pH extremes requires the recipes to be optimized (in terms of gradient linearity) for each desired level of beta av.     

Electrofocusing of acid phosphatases from frog liver, using an immobilized pH gradient

Isoelectric focusing on carrier ampholyte-containing immobilized pH gradient gels was applied (i) to gels submerged in silicone oil on a Peltier cooled apparatus, (ii) to the separation of the higher molecular weight (HMW, Mr 140,000) and the lower molecular weight (LMW, Mr 38,000) acid phosphatases (AcPases) from frog livers. (i) Electrofocusing was conducted on gels submerged under silicone oil cooled and stirred on a Peltier-thermoregulated horizontal gel support plate. This procedure aimed at a) improving the temperature control of the gel by direct contact of coolant with the gel surface, and thus at being able to focus at the maximal field strength and consequently highest resolution; b) preventing evaporation from the gel and c) excluding atmospheric carbon dioxide. Silicone oil submersion did not abolish water loss from the gel into the electrolyte strips during isoelectric focusing, or a rippled gel surface. Absence of water exudation on the ripples noted previously by Atland [1] was observed. (ii) The electrofocusing of AcPases on immobilized pH gradients yielded patterns which remained stationary as a function of time, by contrast to previous analyses on carrier ampholyte generated pH gradients. The total number of enzymatically active components found in the enzyme preparations from different stages of purification and in the isolated HMW and LMW AcPases was 18. The HMW and LMW AcPases focused in characteristic pH ranges and exhibited qualitative and quantitative pattern differences. Their band patterns add up to that of a crude preparation containing both enzymes. Neither polyacrylamide gel electrophoresis (PAGE) at any nondenaturing pH, nor isoelectric focusing in carrier ampholytes with pattern changes due to the pH gradient drift were able to yield that result.