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.     

Improved resolution with one-dimensional polyacrylamide gel electrophoresis: myofibrillar proteins from typed single fibers of human muscle

Standard procedures for one-dimensional discontinuous sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and silver staining were modified to give more effective separation and an improved resolution of human skeletal muscle proteins. In this system, an electrophoresis buffer composed of 100 mM L-isoleucine, 25 mM Tris base, and 0.1% SDS was used. The separating gel consisted of 16% acrylamide with N,N'-methylenebisacrylamide as a crosslinker (1:23), 0.4% SDS, 1.5 M Tris-HCl, pH 8.8. By the present procedure, the slow and the fast forms of myosin light chains (LCs, LCf) and other contractile proteins from human muscle could be better separated. The silver stain is based on a combination of methods previously described. The modified method requires a small fragment of a single fiber to observe as few as 10 ng of myofibrillar muscle proteins. The described simplifications made it possible to assay and compare up to 40 single fibers in the same electrophoretic run. Improved separation of other proteins migrating at basic pH could be achieved by a similar approach.     

Free-flow electrophoresis of proteins in phenol-containing solvents at various pH values

1. Several proteins were found to migrate when subjected to free-flow electrophoresis in buffered phenol-ethanediol-water (3:2:3, w/v/v) solvent mixtures. Mobility of these proteins changed with changing pH (apparent) values of this medium. A pH value of zero mobility for each individual protein could be estimated. 2. Founded on these observations, a high-voltage electrophoresis method in free-flowing buffer films was worked out. The method as presented here was particularly suitable for the separation of proteins on a preparative scale. Application of this and other protein fractionation techniques in dissociating media for the investigation of structural and other insoluble proteins was discussed.     

Peptide mapping of basic proteins by proteolysis in acetic acid/urea-minislab polyacrylamide gels

A method to obtain peptide maps of basic proteins on acetic acid/urea (AU) -polyacrylamide minislab gels is presented. Basic proteins such as the histones are digested with Staphylococcus aureus V8 protease in the stacking gel (pH 4) of an AU-polyacrylamide minislab gel. As the peptides are resolved in the AU minislab gel on the basis of charge and size, it is possible to separate peptides containing modified amino acids from the unmodified, parent peptide. The peptide(s) containing the modified residue may be identified following electrophoresis on a second-dimension sodium dodecyl sulfate-polyacrylamide minislab gel. This procedure will be useful for comparing histone variants and for the study of histone modifications.     

Sequential two-dimensional and acetic acid/urea/Triton X-100 gel electrophoresis of proteins

A method is described which combines the resolving power of two-dimensional gel electrophoresis with that of acetic acid/urea/Triton X-100 gel electrophoresis, avoiding the necessity of eluting protein from the gels at any step of the procedure. The combination of electrophoretic separation on the basis of charge, mass, and hydrophobic properties of the proteins has the potential of resolving modified forms and isoforms present in very complex protein populations. The technique can be used for analytical purposes, or it may be scaled up to yield microgram amounts of highly purified proteins. The resolution obtained by tandem application of nonequilibrium pH gradient electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and polyacrylamide gel electrophoresis in the presence of nonionic detergent was evaluated using crude nuclear proteins of the nematode Caenorhabditis elegans.     

Electrophoresis of proteins on filter paper

A simplified procedure for filter paper electrophoresis is described in which disturbing factors such as evaporation, heating, buffer concentration gradients, and pH changes in the electrode vessels were reduced to a minimum. Artificial mixtures of highly purified proteins could be separated and the components isolated. The application of the method to a variety of studies on serum proteins is demonstrated. Protein concentration in paper segments was determined by two different methods of protein estimation. Curves were obtained showing the same five major peaks for normal serum as found by the classical methods of free electrophoresis. Comparisons were made of the areas of the various components under the curves obtained with the different methods. Two dimensional electrophoresis was applied to serum and serum components. It proved of value in demonstrating the heterogeneity of fractions such as the gamma-globulin of serum. The polysaccharide dextran was used as an index of the extent of electro-osmotic flow during the course of the various experiments. The ratio of the distance of electroosmotic flow and the distance of protein migration was shown to be constant for a given type of paper. For serum albumin on Munktell 20 paper this ratio was 0.35. A formula for mobilities applicable to liquid in a highly porous supporting medium is presented. Mobility values for human serum albumin at various pH levels on paper showed approximate agreement with those obtained in free solution giving a similar isoelectric point.     

Gradiflow as a prefractionation tool for two-dimensional electrophoresis

The Gradiflow trade mark, a preparative electrophoresis instrument capable of separating proteins on the basis of their size or charge, was used to separate whole cell lysates, prepared from bakers yeast (Saccharomyces cerevisiae) and Chinese snow pea seeds (Pisum sativum macrocarpon), into protein fractions of different pH regions. Both broad and narrow range (with a difference of approximately 1 pH unit) pH fractions were obtained. Analysis of the protein fractions by isoelectric focusing gels and two-dimensional (2-D) polyacrylamide gel electrophoresis indicated minimal overlap between the pH fractions. Further, when the prefractionated acidic samples were analyzed on pH 4-7 immobilized pH gradient 2-D gels, improved resolution of the proteins within the chosen pH region was achieved compared to the unfractionated samples. This study demonstrates that the Gradiflow could be used as a preparative electrophoresis tool for the isolation of proteins into distinct pH fractions.     

Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome analysis strategy

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).     

Isolation and characteristics of surface proteins from Streptococcus group A

Cell wall surface proteins of group A Streptococcus type 29 were extracted with 1 M hydroxylamine pH 6.0. The purification procedure included fractionation with ammonium sulfate and gel filtration on Sephadex G-150. SDS polyacrylamide gel electrophoresis revealed a number of proteins (approximately 20) with molecular mass of 70 kD; the difference in Mr between the proteins was 5-10 kD. Isoelectrofocusing demonstrated that the proteins are either acid (pI = 3.7) or weakly alkaline (pI = 7.7). Possible reasons for the heterogeneity of Streptococcus cell wall surface proteins are discussed.     

Polyacrylamide gel-urea electrophoresis of cereal prolamins at acidic pH

An electrophoretic procedure is described for vertical polyacrylamide gel-urea electrophoresis of cereal prolamins. The polyacrylamide gel is directly polymerized in potassium lactate buffer, pH 3.6, with an ammonium persulfate-silver nitrate system of catalysts, which enables one to control the polymerization of acrylamide. Electrophoresis is performed in aluminum lactate buffer, pH 3.6. Thus proteins are separated in a discontinuous system of buffers, which allows sample concentration at the beginning of the electrophoresis.Because the system acts in a very dissociating medium (6 m urea), any type of prolamins (e.g., gliadin, secalin, hordein, avenin, or zein) can be simultaneously analyzed and compared on the same gel slab. On the other hand, as is shown with barley prolamins, improved resolution is obtained, without any sample-reducing requirement during the same run, for both typical hordeins (B- and C-hordeins) and fast-moving low-molecular-weight proteins (A-polypeptides).     

Chromium-associated 32P-labeling of proteins

The incubation of proteins with chromium (Cr3+ or Cr6+) in the presence of 32P ([gamma-32P]ATP or H3(32)PO4) at room temperature for 10-30 min resulted in the labeling of these proteins with 32P. The 32P-labeled proteins could be separated by SDS-polyacrylamide gel electrophoresis and identified by exposure to X-ray film. The characteristics of this procedure included: the optimal chromium concentration was 100 microM; the minimum requirement of each protein was 1 microgram; the optimal pH value was between 6 and 8; metal ions such as V5+, Mn2+ and Fe3+ strongly inhibited the effect of chromium, whereas Ca2+ and Mg2+ had little effect. It was concluded that chromium binds to the proteins and forms a complex with 32P to achieve the 32P-labeling of the proteins. This technique can be applied for the rapid preparation of 32P labels on protein markers for gel electrophoresis and for the identification of unknown protein species.     

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.     

Purification and characterization of biotin-binding protein II from chicken oocytes.

BBP-II, the major biotin-binding protein from chicken oocytes, was purified 12,000-fold with a 22% yield. The purification procedure includes butan-1-ol extraction of yolk lipids, phosphocellulose chromatography of the water-soluble proteins, DEAE-cellulose chromatography at pH 7.4 and hydroxyapatite column chromatography. Final purification was obtained by using a second DEAE-cellulose column chromatography at pH 6.0. BBP-I activity separated from BBP-II activity during elution from the first DEAE-cellulose column. Purified BBP-II was homogeneous on both polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis under conditions that would detect a 1% impurity. The subunit Mr determined from SDS/polyacrylamide-gel electrophoresis was 18,200 (72,600 for tetramer), which compares favourably with an Mr value of 17,300 (69,100) calculated from the amino acid analysis. A single precipitin line formed when rabbit antiserum to the protein was directed against a crude chicken egg-yolk sample. BBP-II purified by this procedure lacked carbohydrate and phosphate, was stable indefinitely when frozen, and was quite stable at room temperature. The N-terminal amino acid sequence showed polymorphism at three positions in the first 23 residues and was about 45% identical with the N-terminal 22 residues of avidin. Antiserum to BBP-II cross-reacted with BBP-I and similar proteins in the yolk of eggs from various birds and alligator as judged by immunodiffusion and enzyme-linked immunosorbent assays. No cross-reaction was observed with chicken egg-white by either of these methods.     

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the protein, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.     

Separation of poly(ADP-ribosylated) nuclear proteins by polyacrylamide gel electrophoresis at acidic pH and low temperature

A method for the extraction and electrophoresis of poly(ADP-ribosylated) nuclear proteins is described. An extraction method using lithium dodecyl sulfate as detergent at pH 2.4 and room temperature is shown to fully extract nuclear proteins under conditions where full stability of protein-linked polymer is ensured. The polyacrylamide gel electrophoresis is performed again under conditions where full stability is ensured. This work provides a technique whereby misinterpretation of relative ADP ribosylation of nuclear proteins can be avoided.     

Affinity chromatography in the separation of human alpha 1-antitrypsin (alpha 1-AT) and antithrombin-III (AT-III).

The two antiproteases alpha 1-antitrypsin (alpha 1-AT) and antithrombin-III (AT-III) have been purified simultaneously from human plasma. Purification procedure consisted of gel filtration on Sephadex G-200 after initial processing of plasma, followed by ion exchange chromatography on DEAE-Sephadex A50 and DEAE-Cellulose, at a pH of 9.0 and pH 8.3 respectively. The two proteins could not be separated by any of these procedures including a lower pH (7.4) in ion exchange chromatography. Affinity chromatography on heparin-Sepharose separated the proteins since alpha 1-AT did not bind to the matrix. Alpha 1-AT unbound to the heparin-Sepharose was subsequently purified through con A-Sepharose affinity column. The final yield of both the proteins was about 20%. The molecular weight estimated on SDS electrophoresis for AT-III and alpha 1-AT was 63,000 and 50,000, respectively.     

Sample prefractionation with Sephadex isoelectric focusing prior to narrow pH range two-dimensional gels

Due to their heterogeneity and huge differences in abundance, the detection and identification of all proteins expressed in eukaryotic cells and tissues is a major challenge in proteome analysis. Currently the most promising approaches are sample prefractionation procedures prior to narrow pH range two-dimensional gel electrophoresis (IPG-Dalt) to reduce the complexity of the sample and to enrich for low abundance proteins. We recently developed a simple, cheap and rapid sample prefractionation procedure based on flat-bed isoelectric focusing (IEF) in granulated gels. Complex sample mixtures are prefractionated in Sephadex gels containing urea, zwitterionic detergents, dithiothreitol and carrier ampholytes. After IEF, up to ten gel fractions alongside the pH gradient are removed with a spatula and directly applied onto the surface of the corresponding narrow pH range immobilized pH gradient (IPG) strips as first dimension of two-dimensional (2-D) gel electrophoresis. The major advantages of this technology are the highly efficient electrophoretic transfer of the prefractionated proteins from the Sephadex IEF fraction into the IPG strip without any sample dilution, and the full compatibility with subsequent IPG-IEF, since the prefactionated samples are not eluted, concentrated or desalted, nor does the amount of the carrier ampholytes in the Sephadex fraction interfere with subsequent IPG-IEF. Prefractionation allows loading of higher protein amounts within the separation range applied to 2-D gels and facilitates the detection of less abundant proteins. Also, this system is highly flexibile, since it allows small scale and large scale runs, and separation of different samples at the same time. In the current study, this technology has been successfully applied for prefractionation of mouse liver proteins prior to narrow pH range IPG-Dalt.     

Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid-urea polyacrylamide gel

Two-dimensional electrophoresis with immobilized pH gradient (IPG) followed by acetic acid/urea-polyacrylamide gel electrophoresis (AU-PAGE) was developed for the detection of low-molecular weight food allergens. Wheat proteins were used to test the applicability of AU-PAGE for the analysis of food allergens. Isoelectric focusing (IEF) for first dimension was performed with IPG pH 3-10. AU-PAGE was performed as a second-dimensional electrophoresis and high resolution was obtained, especially for proteins below 15 kDa. For immunodetection, the proteins resolved on AU gel were transferred to a polyvinylidene difluoride membrane. The assembly of semidry electroblotting for AU gel was set reversed as for sodium dodecyl sulfate (SDS)-PAGE gel. The electroblotted membrane was immunolabeled with serum from a radio-allergosorbent test-positive individual for wheat to identify allergenic proteins. Protein spots strongly recognized by the patient's serum were chosen for further analysis. Mass spectrometry analysis revealed that these proteins were alpha-amylase/trypsin inhibitors and lipid transfer protein. The system developed in this study was shown to be useful as a standard protocol for the separation of low-molecular weight proteins. Moreover, the IPG strips on which IEF was performed could be used either for SDS-PAGE or AU-PAGE by only changing equilibrating conditions, allowing for a wide range of allergen analysis.     

Analysis of rat serum apolipoproteins by isoelectric focusing. I. Studies on the middle molecular weight subunits.

Analytical isoelectric focusing (IEF) has been applied to the study of the apolipoprotein components of rat serum high density and very low density lipoproteins. The apolipoproteins were separated on 7.5% polyacrylamide gels containing 6.8% urea, with a pH gradient of 4-6. The middle molecular weight range apolipoproteins were identified on IEF gels by the use of apolipoproteins purified by electrophoresis on gels containing sodium dodecyl sulfate (SDS). The A-1 protein focused as 4 to 5 bands from pH 5.46 to 5.82; the A-IV protein and the arginine-rich protein each focused as 4 to 6 bands from pH 5.31 to 5.46. The low molecular weight proteins focused from pH. 4.43 to 4.83 and are the subject of a separate communication. Comparisons of the IEF method with SDS gel electrophoresis, polyacrylamide gel electrophoresis in urea, and Sephadex chromatography are also reported. Additional studies were also carried out that tend to rule out carbamylation or incomplete unfolding of the proteins in the presence of urea as the causes of the observed heterogeneity.     

Isoelectric focusing following by electrophoresis of proteins for visualizing their titration curves by zymogram and immunofixation

After isoelectric focusing followed by electrophoresis at right angles in the same gel slab, it is possible to visualize the titration curve of proteins by zymograms or immunofixation even of an unpurified sample. This information can be very useful for the selection of the proper purification strategy by charge-dependent methods, e.g. ion-exchange chromatography, zone and disc electrophoresis and isotachophoresis. The titration curve also gives information on the stability of the protein as a function of the prevailing pH of the medium, in the pH 3-10 range. A region of instability is found for most proteins in acidic conditions, below pH 4.5, while most proteins are stable in the alkaline pH region, at least up to pH 10. The best method for developing zymograms and immunoprints appears to pH 10. The best method for developing zymograms and immunoprints appears to be the 'sandwich technique', by which a thin agarose slab, cast on an hydrophilic polyester sheet, and impregnated with appropriate reagents, is left in contact with a polyacrylamide gel thin layer used to generate the titration curves.