Ultrathin-layer agar gels: a novel print technique for ultrathin-layer isoelectric focusing of enzymes

A novel, rapid, and simple print technique for ultrathin-layer isoelectric focusing of enzymes consisting of ultrathin-layer agar gels on polyester films is presented. Details for preparation of agar gels and the application for detection of commercial amylase, protease, pectinesterase, and lipase are given.     

A procedure for the immunoblotting of proteins separated on isoelectric focusing gels

A method has been devised for performing Western blot assays on proteins resolved by isoelectric focusing. Electrophoretic transfer of proteins directly from isoelectric focusing (IEF) tube gels to nitrocellulose sheets allowed their immunoassay without conventional second dimension SDS gel electrophoresis. The same method can also be used for IEF slab gels. For the immunostaining of nonmuscle actin isoforms in extracts of cultured cells, the resolution of this technique was much improved over that of Western blots of two-dimensional gels.     

High resolution two-dimensional electrophoresis of basic as well as acidic proteins.

A previously described two-dimensional electrophoresis procedure (O'Farrell, 1975) combined isoelectric focusing and sodium dodecylsulfate slab gel electrophoresis to give high resolution of proteins with isoelectric points in the range of pH 4–7. This paper describes an alternate procedure for the first dimension which, unlike isoelectric focusing, resolves basic as well as acidic proteins. This method, referred to as nonequilibrium pH gradient electrophoresis (NEPHGE), involves a short time of electrophoresis toward the cathode and separates most proteins according to their isoelectric points. Ampholines of different pH ranges are used to optimize separation of proteins with different isoelectric points. The method is applied to the resolution of basic proteins with pH 7–10 Ampholines, and to the resolution of total cellular proteins with pH 3.5–10 Ampholines. Histones and ribosomal proteins can be readily resolved even though most have isoelectric points beyond the maximum pH attained in these gels. The separation obtained by NEPHGE with pH 3.5–10 Ampholines was compared to that obtained when isoelectric focusing was used in the first dimension. The protein spot size and resolution are similar (each method resolving more than 1000 proteins), but there is less resolution of acidic proteins in this NEPHGE gel due to compression of the pattern. On the other hand, NEPHGE gels extend the range of analysis to include the 15–30% of the proteins which are excluded from isoelectric focusing gels. The distribution of cell proteins according to isoelectric point and molecular weight for a procaryote (E. coli) was compared to that of a eucaryote (African green monkey kidney); the eucaryotic cell proteins are, on the average, larger and more basic.     

Gel gradient electrophoresis, isoelectric focusing and two-dimensional techniques in horizontal, ultrathin polyacrylamide layers

An ultrathin layer, horizontal polyacrylamide gel system for electrophoresis, isoelectric focusing and two-dimensional techniques is described. Gel slabs 240 micron thin for unidimensional, or 360 micron thin for two-dimensional runs are cast on cellophane foils as support. The sample is loaded in pockets pre-cast in the gel (2--3 microliter size) or in trenches for two-dimensional experiments. The second dimension is routinely performed in concave exponential gel gradients, spanning an acrylamide concentration from 4% to 22.5%. The sensitivity with the common Coomassie Blue stain is very high, well below 0.1 microgram protein/band. Zymogram detections can be developed within a few minutes, thus retaining the band sharpness of the focused zones or of the bands separated in pore gradient electrophoresis. Sample handling, staining and destaining and gel drying and storage are greatly simplified and performed in a fraction of the time needed for conventional, thick gels in the 1-2 mm thickness range.     

Isoelectric focusing in thin-layer acrylamide geis: an improved method for sample application.

Various procedures for applying protein solutions for isoelectric focusing in thin layers of polyacrylamide gels have been described (1). In the most widely used procedure, pieces of filter paper soaked in the solution are laid on the gel surface. The major drawback of this very elegant and simple method is that some proteins will adsorb to the paper. The procedure described here, in which the samples to be analyzed can be applied directly on the gel by means of a specially designed applicator, permits a quantitative approach of isoelectric focusing separation.     

A comparison of silver staining methods for detecting proteins in ultrathin polyacrylamide gels on support film after isoelectric focusing

The application of silver staining methods to the detection of proteins on ultrathin isoelectric focusing gel systems requires the optimization of many steps in the procedure in order to obtain reproducible staining of proteins with acceptable levels of background. Three different methods which have been reported for detecting proteins by silver staining in sodium dodecyl sulfate-polyacrylamide gel systems were investigated. A major problem with staining ultrathin isoelectric focusing gels was found to be surface staining that was associated with gels cast on support films. A modification of the method of Poehling and Neuhoff (H.-M. Poehling and V. Neuhoff, 1981, Electrophoresis 2, 141-147) was found to give the best results.     

Procedure for simultaneous phenotyping of genetic variants in cow's milk by isoelectric focusing*

A method is described which allows the simultaneous separation of all polymorphic protein fractions in cow's milk in one single run. The separation could be achieved by isoelectric focusing in ultrathin-layer polyacrylamide gels. The method is especially suitable for screening purposes because it combines low costs, high resolution and short separation time.     

Poly-N-acryloyl-Tris gels as anticonvection media for electrophoresis and isoelectric focusing

We describe in detail the synthesis of an acrylic monomer, N-acryloyl-tris(hydroxy-methyl)aminomethane (NAT), which was successfully used for the preparation of gels for electrophoresis and isoelectric focusing. The polymerization kinetics and transparency of the poly(NAT) gels crosslinked by N,N'-methylenebisacrylamide (Bis) are also shown. Poly(NAT)-Bis gradient (4-24%) gel resolves proteins according to their size. The exclusion limit of this gel is slightly over 3 X 10(6), which is more than threefold higher than the exclusion limit of the polyacrylamide gradient gel of the same concentration. The gel made of 6% NAT and 3% Bis represents a suitable matrix for isoelectric focusing. These results demonstrate that poly(NAT)-Bis gels could be advantageously used in those applications where the extensive sieving by the polyacrylamide matrix is not desir desirable.     

Polyacrylamide gel electrophoresis: recovery of non-stained and stained proteins from gel slices

Following electrophoresis or isoelectric focusing in gels of polyacrylamide the protein band of interest is cut out and placed above a sucrose gradient column, containing carrier ampholytes (Pharmalyte). By electrophoresis, isoelectric focusing or displacement electrophoresis the proteins migrate out of the gel slice and into the isoelectric focusing column for concentration and further purification. From this column, the proteins can be withdrawn and their isoelectric points determined. Even after staining with Coomassie Brilliant Blue at least some proteins can be recovered by this technique and used for further analyses, for instance amino acid determinations. The focusing in a pH gradient by carrier ampholytes can be replaced by an electrophoresis in a conductivity gradient column. However, in comparison with isoelectric focusing, this concentration technique has the drawback of not permitting further purification of the eluted protein.     

Procedure for simultaneous phenotyping of genetic variants in cow's milk by isoelectric focusing

A method is described which allows the simultaneous separation of all polymorphic protein fractions in cow's milk in one single run. The separation could be achieved by isoelectric focusing in ultrathin-layer polyacrylamide gels. The method is especially suitable for screening purposes because it combines low costs, high resolution and short separation time.     

Rapid isoelectric focusing in a vertical polyacrylamide minigel system

A rapid method is described for the resolution of proteins employing isoelectric focusing in a vertical polyacrylamide minigel system. Isoelectric focusing can be performed in only 3 h, utilizing low voltage, under either native conditions or denaturing conditions in the presence of 8 M urea. The procedure permits the application of larger sample volumes containing more protein than other isoelectric focusing procedures, and provides the additional advantages of slab gels over tube gels for analytical purposes. The procedure is also well adapted for use in two-dimensional electrophoretic techniques, making it possible to complete a two-dimensional gel in 1 day.     

Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients

A reproducible high-resolution protein separation method is the basis for a successful differential proteome analysis. Of the techniques currently available, two-dimensional gel electrophoresis is most widely used, because of its robustness under various experimental conditions. With the introduction of narrow range immobilized pH gradient (IPG) strips (also referred to as ultra-zoom gels) in the first dimension, the depth of analysis, i.e. the number of proteins that can be resolved, has increased substantially. However, for poorly understood reasons isoelectric focusing on ultra-zoom gels in the alkaline region above pH 7 has suffered from problems with resolution and reproducibility. To tackle these difficulties we have optimized the separation of semipreparative amounts of proteins on alkaline IPG strips by focusing on two important phenomena: counteracting water transport during isoelectric focusing and migration of dithiothreitol (DTT) in alkaline pH gradients. The first problem was alleviated by the addition of glycerol and isopropanol to the focusing medium, leading to a significant improvement in the resolution above pH 7. Even better results were obtained by the introduction of excess of the reducing agent DTT at the cathode. With these adaptations together with an optimized composition of the IPG strip, separation efficiency in the pH 6.2-8.2 range is now comparable to the widely used acidic ultra-zoom gels. We further demonstrated the usefulness of these modifications up to pH 9.5, although further improvements are still needed in that range. Thus, by extending the range covered by conventional ultra-zoom gels, the depth of analysis of two-dimensional gel electrophoresis can be significantly increased, underlining the importance of this method in differential proteomics.     

A universal method for two-dimensional polyacrylamide gel electrophoresis of membrane proteins using isoelectric focusing on immobilized pH gradients in the first dimension.

Two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension, initially applied for the separation of soluble and total cellular proteins, has been extended to the analysis of membrane proteins. We show that the usual procedures lead to artifacts and irreproducible results due to aggregation and precipitation of proteins and protein-phospholipid complexes during isoelectric focusing (first dimension) and sodium dodecyl sulfate (SDS) gel electrophoresis (second dimension). Optimized solubilization procedures for hydrophobic membrane proteins are presented and the use of dilute samples is shown to be essential to overcome the major problems in isoelectric focusing. Increased volumes of samples dissolved in rehydration buffer are applied by direct rehydration of dry immobilized pH gradient (IPG) gels. Isoelectric focusing in 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) without urea gives good results as does 2% Nonidet-P40 with 8 M urea. Heat denaturation should be avoided. An optimized equilibration procedure for IPG gel strips in SDS sample buffer prior to separation in the second dimension was developed that minimizes loss of proteins and results in high-resolution two-dimensional electropherographic maps with a minimum of streaking. The gel strips are partially dehydrated at 40 degrees C and shortly reswollen in situ on the SDS slab gel in SDS-sample buffer containing agarose.     

A gel transfer tank for immunoblotting and its application for analysis of nuclear protein antigens

The design of a gel transfer tank for immunoblotting is described. It is simple and cheap to make, provides a uniform field and uniform transfer over the whole area of the gel, and can easily be adapted for use with any size of gel. It has been used for transfer of proteins from both sodium dodecyl sulfate-polyacrylamide and isoelectric focusing gels to nitrocellulose membranes and its application to the analysis of nuclear proteins is described.     

A multiple high-resolution mini two-dimensional polyacrylamide gel electrophoresis system: imaging two-dimensional gels using a cooled charge-coupled device after staining with silver or labeling with fluorophore.

A multiple mini two-dimensional electrophoretic method which results in three two-dimensional protein spot patterns being positioned side by side in an individual gel has been developed. Preparation time has been minimized by employing disposable capillary tubes for the isoelectric focusing gels and reducing the number of second-dimensional gels required. Commercially available vertical slab units were used for the second-dimensional electrophoresis. The protein spot patterns were visualized either by staining the second-dimensional gel with silver or fluorescently labeling the focused proteins while present in the isoelectric focusing gel and subsequently electrophoresing them into the second-dimensional gel. The fluorescently labeled second-dimensional gel was imaged while still present in the glass mold immediately following electrophoresis. Two fluorophores were compared: 2-methoxy-2,4-diphenyl-3(2H)-furanone and 5-(4,6-dichlorotriazin-2-yl)aminofluorescein hydrochloride. A rapid imaging system based on a cooled charge-coupled device was used to view both the silver-stained and fluorescently labeled two-dimensional spot patterns. The sensitivity of detection of protein spots in the mini two-dimensional gels was similar for the two types of fluorescently labeled gels and the silver-stained gels.     

Preparative isoelectric focusing in agarose

The use of agarose gels as supporting media for flat-bed preparative isoelectric focusing was applied to the fractionation of serum proteins in the pH range 3.5–6, and red cell hemolysates in the pH range 3–8. The agarose gels are easy to prepare, give linear pH gradients, and do not appear to produce molecular sieving effects. Up to 1 g serum proteins can be loaded on the gels, with recoveries between 68 and 82%. Nucleoside phosphorylase from red cell lysates was recovered with 76% yield, indicating that no appreciable denaturation of this enzyme had occurred. Preparative isoelectric focusing in agarose gels provides a useful alternative to existing techniques of preparative isoelectric focusing in sucrose gradients or granulated gels.     

Charge-dependent staining of proteins after electrophoretic separation in polyacrylamide gels

A staining procedure is described which allows for the identification of basic and acidic proteins after gel electrophoresis. This includes electrophoresis of sodium dodecylsulfate (SDS) membrane proteins not accessible to isoelectric focusing as a means of charge-dependent separation. Nonfixative charge-dependent staining can be used for detection of proteins after separation in gels, when further investigation of the intact protein is desired.     

Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution.

The development of a dedicated two-dimensional gel electrophoresis system is described that provides superior performance in terms of high resolving power and enhanced gel-to-gel reproducibility. Isoelectric focusing is performed in a 1-mm capillary tube with a 0.08-mm thread, optimized for this application, incorporated along its length prior to polymerization of the gel matrix. The isoelectric focusing gel is 4% T, 2.6% C to minimize sieving of proteins and promote adhesion of the gel to the thread. The thread incorporated in the isoelectric focusing matrix prevents gel stretching and breakage during its application to the second dimension. An optimum ampholyte pH range has been defined based on 1600 polypeptides present in a transformed fibroblast cell lysate and verified using a variety of other cell types. The length of time required to complete an electrophoretic separation in the second dimension was found to depend on buffer conductivity establishing the importance of high quality electrophoresis grade reagents devoid of contaminating salts. To ensure reproducibility of electrophoretic separations, it is critical to maintain a strict control of temperature during the second dimension separation. This prevents altered migration of some polypeptides relative to neighboring polypeptides that have constant Rfs over a broad temperature range. It was also determined that to obtain the maximum information from a complex protein mixture it is critical to use a large format 22- x 22-cm two-dimensional electrophoretic system. Using the optimized two-dimensional electrophoretic system and computerized gel analysis, it was determined that molecular weight estimates of polypeptides differed by approximately 350 daltons between gels, while isoelectric point estimates differed by approximately 0.03 pH units between gels. Using the two-dimensional electrophoresis system described, approximately 1000 polypeptides can be routinely detected from silver-stained 10% polyacrylamide gels or 1600 polypeptides from autoradiographs of 35S-methionine-labeled polypeptides.     

Over two hundred polypeptides resolved from the human erythrocyte membrane

A modification of O'Farrell's method of two-dimensional polyacrylamide gel electrophoresis has allowed for the resolution of erythrocyte membranes showing up to 200 individual components. Data is presented which indicates that this protein heterogeneity is not produced by artifactual protein-protein aggregation, ednogenous protease activity of secondary charge modification. Similar patterns are obtained when the samples are added to the unpolymerized isoelectric focusing gel, and isolated and stored in protease inhibitor. Individual spots could be eluted off of stained gels, resolubilized under extreme detergent solubilization conditions and run on one-dimensional gels; these run as sodium dodecyl sulfate in the solubilization procedure. The method chosen for solubilization prior to isoelectric focusing appears to cause selective aggregation of all or most of the spectrin and band 3 proteins. This further allows for excellent resolution of more components.     

Protein staining and pH gradient determination on the same gel in isoelectric focusing.

The apparent isoelectric point of a component focused on polyacrylamide gels is normally estimated by extrapolating a pH gradient determined on one gel to another gel which has been stained for protein in order to locate the position of the component (1). The pH gradient is determined by slicing the gel transversely and reading the pH of the eluate after soaking the segments for 1–2 hr in a small amount of degassed water. It is assumed that the gradients in both gels are identical. Alternatively, an antimony microelectrode has been used to measure pH gradients directly in unsectioned gels (2). Similar techniques have been applied to polyacrylamide gel slabs and are reviewed by Vesterberg (3). Righetti and Drysdale (4) have recently reviewed these and other aspects of isoelectric focusing in gels.I report here a very precise method for the determination of a protein “isoelectric point” that can be accomplished with a single gel. The technique is demonstrated with yeast phosphoglycerate kinase and the very low density lipoprotein (VLDL) fraction from human plasma.