Two-dimensional Affinity Electrophoresis Research Articles

Identification of two phosphorylated species of β-catenin involved in the ubiquitin-proteasome pathway by using two-dimensional Phos-tag affinity electrophoresis

We recently reported a neutral-pH gel system buffered with 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol hydrochloride (Bis-Tris-HCl) for use in Zn2+-Phos-tag sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for advanced profiling of protein phosphorylation. In the current study, we extended the utility of Zn2+-Phos-tag SDS-PAGE with the Bis-Tris-HCl buffer system to a detailed analysis of phosphorylated β-catenin, which is closely involved in the ubiquitin-proteasome pathway. The Phos-tag-based approach, followed by Western blotting with an anti-β-catenin antibody, allowed us to assign nine phosphorylated species of β-catenin produced in complicated signaling pathways of cultured HEK293 and SW480 cells. Two-dimensional image coupling with normal Laemmli’s SDS-PAGE as the first dimension gave more detailed information, not only on the phosphorylation of β-catenin, but also on the phosphorylation-dependent polyubiquitination by visualizing multiple ubiquitinated forms derived from two phosphorylated species of β-catenin in lactacystin-treated HEK293 cells. We identified two distinct phosphorylated species of β-catenin that are responsible for polyubiquitination. The first contains phosphorylated residues at S33, S37, T41, and S45, and the second contains these sites and an additional phosphorylated residue at S675. The profiling of double post-translational modifications of β-catenin is consistent with the widely accepted phosphorylation-dependent ubiquitination model in the absence of a Wnt signal.

Further resolution of α-fetoprotein glycoforms by two-dimensional isoelectric focusing and lectin affinity electrophoresis

Human alpha-fetoprotein (AFP) from serum of patients with cirrhosis and hepatocellular carcinoma (HCC) was separated into several bands by IEF and by erythroagglutinating phytohemagglutinin (E-PHA) affinity electrophoresis. These AFP bands were directly compared in 2-D IEF and E-PHA affinity electrophoresis. IEF of serum AFP was run in 1% agarose IEF gel with 3% Pharmalyte 4.5-5.4. After IEF, a part of the gel was stained for AFP and another part of the gel corresponding to the area of separated AFP bands was cut in 1 mm x 39 mm along the focused direction and transferred to a trough in 1% agarose gel with 0.3 mg/mL E(4)-PHA for second-dimensional affinity electrophoresis. Separated 2-D AFP spots were visualized by antibody-affinity blotting and identified by combining the systems of Johnson et al.. (Johnson, P. J., Ho, S., Cheng, P., Chan, A. et al.., Cancer 1995, 75, 1663-1668) for AFP-I-+IV and of Taketa et al.. (Taketa, K., Ichikawa, E., Taga, H., Hirai, H., Electrophoresis 1985, 6, 492-497) for AFP-P1-5. AFP-P2, the major AFP glycoform, was composed of AFP-I, AFP+I, and AFP+II; AFP-P3, a nonspecific monosialo-AFP, was composed of AFP+II; AFP-P4, HCC-specific monosialo-AFP, was composed of AFP+II, AFP+III, and AFP+IV; and malignancy-related AFP-P5 was composed of AFP+I and AFP+II. Monosialo-AFP (AFP+II) was recovered in all the glycoforms of AFP-P2, -P3, -P4, and -P5; thus, AFP-P4 is more specific to HCC than monosialylated AFP+II.

Characterization of sugar chain structures of human alpha-fetoprotein by lectin affinity electrophoresis. ibarahp@oka.urban.ne.jp.

Alpha-Fetoprotein (AFP) glycoforms, defined as AFP with different chemical structures of carbohydrate, were analyzed by affinity electrophoresis with several lectins of known specificities against complex-type oligosaccharides. Serum AFP samples from cord blood on full-term delivery and from patients with hepatocellular carcinoma and extrahepatic malignancies including gastrointestinal tumors and yolk sac tumors were used. Two-dimensional lectin affinity electrophoresis and also lectin affinity chromatography coupled with lectin affinity electrophoresis were employed. More than ten AFP glycoforms were identified or characterized using the above-mentioned AFP samples. Known specificities of the lectins against complex-type oligosaccharides were refined or their additional specificities were found in this study. Lectin appeared to have specificity against carbohydrates by recognizing not only specific residues but also the whole carbohydrate molecule containing the residues, resulting in differential affinities for the lectin.

Analysis of Protein–Protein Interaction by Two-Dimensional Affinity Electrophoresis

Analysis of Protein–Protein Interaction by Two-Dimensional Affinity Electrophoresis

Effects of carrier and hapten array on the production of anti-hapten antibodies analyzed by two-dimensional affinity electrophoresis.

Effects of a carrier, bovine serum albumin (BSA) and a hapten array, fluorescein isothiocyanate-conjugated dextran (FITC-DEX), on the production of anti-FITC antibodies in BALB/c mice were analyzed by two-dimensional affinity electrophoresis (2-D AEP). The mice were immunized with FITC-BSA, followed by immunizations with FITC-BSA, saline, BSA, human serum albumin (HSA), fluorescein (Flu), and FITC-DEX. The heterogeneity and quantity of anti-FITC antibodies were increased markedly during the second and third immunization with FITC-BSA. However, the production of anti-FITC antibodies with low affinity to FITC was suppressed by the second and third immunization with the carrier protein of BSA. The suppression of anti-FITC antibodies by immunization with BSA may be due to the major histocompatibility complex (MHC)-mediated competition through antigen processing and the clonal expansion of BSA-specific B cells because similar suppression was induced by the immunization with HSA. The hapten array of FITC-DEX generally suppressed the production of anti-FITC antibodies; however, immunization with free hapten of Flu did not affect the production significantly. This may indicate that cross-linking of anti-FITC antibodies on the surface of specific B cells via the binding of hapten array is required for the suppression.

A Putative Sugar-binding Protein in the Chemosensory Organ of the Fruit Fly, Drosophila melanogaster.

A single chemosensillum usually includes five sensory cells, and one of them is traditionally called as the sugar receptor cell. We have collected the legs and wings of the fruit fly, Drosophila melanogaster, using several grades of sieve and here demonstrate the presence of the putative pyranose-site protein by a two-dimensional affinity electrophoresis method.

Difference in Hemoglobin-Binding Ability of Polymers Among Haptoglobin Phenotypes

Langlois and Delanghe (1) described several functional differences between haptoglobin phenotypes in this journal. They reviewed the functional properties of haptoglobin phenotypes such as ability for hemoglobin (Hb) binding, antioxidative capacity, and inhibition of prostaglandin synthesis, which were strong in Hp-1, intermediate in Hp2–1, and weak in Hp2–2. However, we do not agree with their results about the ability for Hb binding. Hp2–1 and Hp2–2 phenotypes form many polymers, and analysis of the functional properties of each of the isolated polymer proteins had been very difficult (2). We designed new analytical methods such as crossed Hb electrophoresis (3) and two-dimensional affinity electrophoresis, using Hb (4) to identify the Hb-binding ability of each of the haptoglobin polymer proteins of Hp2–1 and Hp2–2 phenotypes. From the results of crossed Hb electrophoresis, we found that a haptoglobin molecule combined with equimolar amounts of Hb to form at least a hexamer. We confirmed by two-dimensional affinity electrophoresis that the larger polymer of the Hp2–2 phenotype had a lower affinity to Hb, but a Hp2–1 polymer of very … aAuthor for correspondence.

Analysis of the affinity of each haptoglobin polymer for hemoglobin by two-dimensional affinity electrophoresis

Using polyacrylamide gel electrophoresis we have often encountered a decrease in the high molecular weight polymers of haptoglobin 2-1 in the serum of patients after transfusion and with hemolytic diseases. Using two-dimensional affinity electrophoresis we have attributed this phenomenon to the high affinity of high molecular weight polymers of the haptoglobin 2–1 for hemoglobin.

Estimation of the affinity between haptoglobin and hemoglobin by two-dimensional affinity electrophoresis.

Estimation of the affinity between haptoglobin and hemoglobin by two-dimensional affinity electrophoresis.

Immune response to different doses of a hapten of fluorescein isothiocyanate analyzed by two-dimensional affinity electrophoresis.

The immune response to different doses of a hapten of fluorescein isothiocyanate (FITC) in BALB/c mice was analyzed by two-dimensional affinity electrophoresis (2-D AEP). The mice were immunized with different doses (0.5 microgram, 5 micrograms, 50 micrograms, 500 micrograms and 2.5 mg) of FITC-conjugated bovine serum albumin (BSA). The antibodies to FITC bovine serum albumin (BSA) were separated into a large number of IgG spots due to differences in their isoelectric points (pI) and binding affinity to FITC ligand immobilized in the gel. The IgG spots, showing identical affinity to the ligand but different pI, have been considered as an IgG family. The affinity and quantity of IgG families changed with the increase in FITC-BSA dosage. With a low dose (5 micrograms) most of the families showed high affinity (Kd < 1 microM < Kd < 50 microM) and low affinity (Kd > 50 microM) antibodies were produced. The increase of FITC-BSA up to 500 micrograms markedly increased the quantity of IgG spots showing a variety of affinity to FITC. However, 2.5 mg FITC-BSA did not increase the quantity and heterogeneity of IgG spots significantly. The changes in the heterogeneity and quantity of anti-FITC antibodies and the subclass switch were observed over the course of immunization. The heterogeneity and the quantity of IgG1, IgG2b and IgG3 antibodies increased markedly during the first and the second immunization, whereas an increase in the heterogeneity and the quantity of IgG2a antibody was observed in the third immunization. This suggests that the subclass switch to IgG1, IgG2b and IgG3 and the somatic mutation of IgG1, IgG2b and IgG3 occur during the first and the second immunization, but the subclass switch to IgG2a and the somatic mutation of IgG2a seem to occur later than that of the other IgG subclasses.

Affinity electrophoretic determination of oligosaccharide specificity of Butea monosperma agglutinin.

The oligosaccharide specificity of newly isolated Butea monosperma agglutinin (BMA) was determined by two-dimensional lectin affinity electrophoresis of alpha-fetoprotein (AFP) with concanavalin A, lentil lectin, erythroagglutinating phytohemagglutinin and Allomyrina dichotoma lectin, of which the specificities to known AFP oligosaccharides had been established. Effects of neuraminidase treatment on the reactivities of AFP to the lectins were also studied. The results indicated that BMA had the highest affinity for the exposed Gal residues of nonreducing termini of biantennary complex-type oligosaccharides, and that the affinity was reduced to zero in the following order by the presence of monosialyl residue of the Man alpha1->3 arm, monosialyl residue of the Man alpha1->6 arm, monosialyl residue of the Man alpha 1->3 arm and bisecting G1cNAc, and disialyl residues. BMA did not recognize Neu5Ac alpha2->6 and Neu5Ac alpha2->3 substitutions of Gal. These characteristics of BMA were shown to be useful in identifying malignancy-associated alteration of AFP sugar chains.

Lectin affinity electrophoresis.

Lectin affinity electrophoresis is a powerful technique to investigate the interaction between a lectin and its ligand. Affinity electrophoresis results from the reduced mobility of a charged species owing to its interaction with an immobile species. In this protocol, a two-dimensional lectin affinity electrophoresis experiment is described that affords separation of oligosaccharides. The first-dimension is composed of a weak, polyacrylamide, capillary tube gel containing a lectin. The example described involves a mixture of fluorescently labeled disaccharides. The mobility of only the lectin-binding disaccharide is reduced affording a separation in the first-dimension. The tube gel is then extruded and placed onto the second-dimension gradient polyacrylamide gel and subjected to electrophoresis. Mobility in the second-dimension is dependent on molecular size and visualization si by fluorescence under transillumination. This method is also applicable, with appropriate modifications, for the separation and analysis of glycopeptides and glycoproteins.

Immune response to a hapten of fluorescein isothiocyanate in a single mouse analyzed by two-dimensional affinity electrophoresis.

Immune response to a hapten of fluorescein isothiocyanate (FITC) in a single BALB/c strain mouse was analyzed by two-dimensional affinity electrophoresis (2D-AEP). Anti-FITC antibodies were induced by immunization with FITC-conjugated bovine serum albumin. The antibodies were separated into a large number of spots of IgG due to differences in their isoelectric points(pI) and binding affinities to the FITC ligand. These spots consisted of IgG families which were composed of several spots having an identical affinity to the ligand but a different pI. The spots were not clearly detected in the antiserum taken on day 7 after the primary immunization, but on day 21 the spots of IgG were clearly detected, with a high diversity and specificity for the ligand. The size and number of IgG spots were markedly increased by the secondary immunization; however, the third immunization did not increase the size and number of IgG spots. The IgG spots of each family were specifically stained with an antimouse IgG subclass antibody. Furthermore, a monoclonal antibody (FL-D6) was separated by 2D-AEP into a single family which consisted of seven IgG1 spots having an identical affinity to FITC but different pIs. Therefore, each of the IgG families of anti-FITC antibodies in the antiserum can be generated by a single clone of anti-FITC antibody-producing cells. The substitution of dextran T2000 or lipopolysaccharide for bovine serum albumin as a carrier for FITC induced much smaller amounts of anti-FITC antibodies with a low diversity but high specificity to FITC.

Affinity maturation of anti-hapten antibodies in a single mouse analyzed by two-dimensional affinity electrophoresis.

Affinity maturation of anti-hapten antibodies in an inbred mouse (BALB/c) was analyzed by two-dimensional affinity electrophoresis (2D-AEP) in which the antibodies specific to a hapten of dinitrophenyl (DNP) group were separated into a large number of IgG families according to the differences in their isoelectric points (pI) and in their affinities for the ligand of DNP immobilized in the gel matrices. Each of the IgG families consisted of several spots showing an identical affinity for the ligand but different pI, and the spots were specifically stained by immunostaining with an anti-murine IgG subclass antibody. These results lead us to the conclusion that each of the IgG families is derived from a single clone of antibody-producing cells specific to DNP. The mass and affinity of the IgG families varied in the course of immunization with DNP-conjugated chicken serum albumin (DNP-CSA). The first injection of DNP-CSA induced a small amount of IgG families showing a wide variety in their affinity for DNP. The second injection induced a large amount of IgG families, especially of families having a medium affinity for DNP. Additional injections, however, did not change the mass or affinity of the IgG families significantly. This suggests that an antigen-induced somatic mutation of the immunoglobulin gene does not occur frequently to mature the affinity of antibodies by the additional injections after the second injection. 2D-AEP enables us to analyze the affinity maturation of antibodies in vivo in a single mouse and to document the subclass switch of the antibody in the course of immunization.

Characterization of E-PHA-reactive alpha-fetoprotein isoforms by two-dimensional lectin affinity electrophoresis.

Erythroagglutinating phytohemagglutinin (E-PHA)-dependent isoforms of human alpha-fetoprotein (AFP) from cord blood were analyzed for their carbohydrate structures by two-dimensional electrophoresis with E-PHA combined with extended agarose gel electrophoresis or with affinity electrophoresis with concanavalin A or Allomyrina dichtoma lectin. By means of neuraminidase and/or beta-galactosidase treatment, AFP-P2 was identified as alpha 2-->6 disialo-AFP, AFP-P3 as having biantennary structures with alpha 2-->6 monosialylated galactose of the Mannose (Man) alpha 1-->6 arm, AFP-P4 as having alpha 2-->6 monosialylated galactose of the Man alpha 1-->3 arm, and AFP-P5 as disialo-AFP with alpha 2-->3 sialylated galactose of the Man alpha 1-->6 antenna with the alpha 2-->6 sialylated galactose of the other antenna. Desialylated AFP with the terminal galactose of the Man alpha 1-->6 antenna with or without the galactose of the other arm also had a migration of AFP-P4, and other hydrolytic intermediates without the terminal galactose of the Man alpha 1-->6 arm with and without the galactose of the other antenna had mobilities of AFP-P3s and AFP-P3, respectively. Thus, the present system of two-dimensional lectin affinity electrophoreses would provide a model for the determination of the sugar chain structure of glycoproteins.

Separation of monoclonal antibodies from antihapten antisera by two-dimensional affinity electrophoresis

A high-resolution two-dimensional affinity electrophoresis (2D-AEP) method was developed, using capillary polyacrylamide gel (PAG) isoelectric focusing in the first and slab PAG affinity electrophoresis in the second direction. Using this method, anti-hapten antibodies were separated into a number of monoclonal antibody [immunoglobulin G (IgG)] families, each of which is composed of several IgG spots having an identical affinity to the hapten but different isoelectric points. 2D-AEP may offer a powerful tool for solving fundamental problems in immunochemistry such as antibody heterogeneity, its hapten binding specificity and antigen-dependent somatic mutation.

Analysis of the interaction between an α(1 → 6)dextran-specific mouse hybridoma antibody and dextran B512 by affinity electrophoresis

Carbohydrates are common environmental antigens. As dextran B512 is composed of a repeating structure of simple antigenic determinants, it is widely used to study the immunochemical properties of immunoglobulins. Two-dimensional affinity electrophoresis patterns of a mouse monoclonal antidextran antibody (35.8.2H; IgG 1, BALB/c) were produced to obtain insights into the microheterogeneity of the monoclonal antibody. The monoclonal antibody was separated into about six spots which had an identical affinity to dextran B512, but differed in their isoelectric points (p I). In addition, the pH dependence of the binding affinity of this antidextran to dextran B512 was examined. By comparing affinities obtained by affinity electrophoresis between weakly basic (pH 9.5) and weakly acidic (pH 3.8) discontinuous buffer systems, the latter showed an affinity about 500 times lower than the former. The change in the affinity was investigated with a continuous pH gradient by an affinity titration curve and was seen to change markedly at about pH 6. This suggests that the histidine at residue 34 in the light-chain CDR1 is largely responsible for the dextran binding.

２次元電気泳動法の活用と問題点 ２次元親和電気泳動法による抗ハプテン抗体の分離

２次元電気泳動法の活用と問題点 ２次元親和電気泳動法による抗ハプテン抗体の分離

Studies on the heterogeneity of anti-hapten antibodies by means of two-dimensional affinity electrophoresis.

The molecular heterogeneity of rabbit anti-hapten antibodies has been investigated by two-dimensional affinity electrophoresis (2D-AEP). Anti-dansyl and anti-arsanilic diazo-antibodies were separated into several hundred IgG spots as in the case of anti-DNP antibodies. They were grouped into a number of monoclonal IgG families. At the beginning of immunization, IgG spots having low pI and low affinity were predominant but one or two weeks after immunization the IgG spots with high affinity and high pI increased. After the second or third immunization, the 2D-AEP patterns became stable and constant. Anti-arsanilic diazo and anti-DNP antibodies exhibited only weak cross-reactivity with other aromatic haptens. In contrast, anti-dansyl antibodies cross-reacted to a considerable degree with DNP-hapten. A few anti-dansyl IgG families which have cross-reactivity with DNP-hapten were separated. Their apparent dissociation constants to the haptens and their affinity ratios were calculated from their 2D-AEP patterns, according to the affinity theory.

Two‐dimensional lectin affinity electrophoresis of α‐fetoprotein: Characterization of erythroagglutinating phytohemagglutinin‐dependent microheterogeneity forms

By means of two-dimensional lectin affinity electrophoresis of human alpha-fetoprotein (AFP) from different sources, AFP bands separated with erythroagglutinating phytohemagglutinin (E-PHA) were further characterized with other lectins of known oligosaccharide specificities. The results with a cord serum AFP revealed that not only AFP-P2 (E-PHA-nonreactive) but also AFP-P4 and P5 (E-PHA-reactive) had affinities for Concanavalin A (Con A) and Allomyrina dichotoma lectin (allo A), indicating that the cord serum AFP has nonbisected biantennary complex-type oligosaccharides with the terminal galactose on Man alpha 1----6 residue sialylated at the C-6, but not C-3, position. On the other hand, the results with a hepatoblastoma (HUH-6 C1-5 cell line) AFP showed that not only AFP-P5 but also AFP-P1 (E-PHA-nonreactive) and P3 (E-PHA-less reactive) had Con A-nonreactive AFP and that AFP-P1 had AFP-A1 (allo A-nonreactive) and AFP-A2 (allo A-less reactive), and AFP-P3 and P4 had AFP-A1s (allo A-nonreactive), as main components, in addition to the spots of cord serum AFP. Most of the E-PHA-dependent bands of AFP were further subdivided with Lens culinaris agglutinin (LCA-A) into LCA-A-reactive, weakly reactive and nonreactive spots. Similar results were obtained with AFP preparations from hepatocellular carcinomas and other malignancies, indicating that the bisected bi-(or tri- and tetra-) antennary sugar chains with the exposed terminal galactose of the Man alpha 1----6 arm as well as those with the C-3 sialylated galactose residues could be expressed in AFP upon malignant transformation.(ABSTRACT TRUNCATED AT 250 WORDS)