Polyacrylamide Gel Electrophoresis System Research Articles

A reference map of human nasopharyngeal squamous carcinoma proteome

In order to conduct a comparative proteomics study of human nasopharyngeal carcinoma (NPC) to understand the molecular mechanisms that participate in the formation of NPC, the two-dimensional gel electrophoresis (2-DE) reference map of human NPC tissue proteome was described. To provide a high level of reproducibility between gels and accurately array each protein expressed in NPC tissue proteome, the two-dimensional polyacrylamide gel electrophoresis system, modified colloidal Coomassie Brilliant Blue staining method and ImageMaster 2D Platinum image analysis software were used. The NPC 2-DE maps show that high quality and good reproducibility of the 2-DE gel pattern was attained. An average total of 1,100 protein spots were separated by 2-DE, visualized by a modified colloidal Coomassie Brilliant Blue staining method. A synthesized 2-DE reference gel was acquired after detailed analysis of the NPC 2-DE gel maps, and 216 medium to high abundant spots were identified as landmark spots of NPC 2-DE gel, which expressed on >75% of gels. To provide an unambiguous identification of the landmark spots in gels, MALDI-TOF, ESI-Q-TOF mass spectrometry and database search were used to identify the proteins expressed in NPC tissue proteome. Between the 216 landmark spots, all proteins were identified with MALDI-TOF at first, 41 of which were identified with both MALDI-TOF and ESI-Q-TOF. All identified proteins were classified in terms of their subcellular localization and physiological function with information from SWISS-PROT and NCBI websites. According to our knowledge this is the first 2-DE reference map of human NPC. This reference map will serve as a basis for further studies of human NPC and the reference map data will be used to expand the proteome database of human NPC, which can be accessed in our website (http://www.xyproteomics.org/).

Blue-native PAGE in plants: a tool in analysis of protein-protein interactions.

Intact protein complexes can be separated by apparent molecular mass using a standard polyacrylamide gel electrophoresis system combining mild detergents and the dye Coomassie Blue. Referring to the blue coloured gel and the gentle method of solubilization yielding native and enzymatically active protein complexes, this technique has been named Blue-Native Polyacrylamide Gel-Electrophoresis (BN-PAGE). BN-PAGE has become the method of choice for the investigation of the respiratory protein complexes of the electron transfer chains of a range of organisms, including bacteria, yeasts, animals and plants. It allows the separation in two dimensions of extremely hydrophobic protein sets for analysis and also provides information on their native interactions. In this review we discuss the capabilities of BN-PAGE in proteomics and the wider investigation of protein:protein interactions with a focus on its use and potential in plant science.

An improved purification procedure for cyclosporin synthetase

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation → gel filtration → hydrophobic interaction chromatography → anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24–29 °C and a pH optimum of 7.6. The native enzyme displayed a p I of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.

A reference map of a human pituitary adenoma proteome.

In order to compare the proteomes from different cell types of pituitary adenomas for our long-term goal to clarify the molecular mechanisms that participate in the formation of pituitary adenoma, and to detect any tumor-related marker for an "early-stage" diagnosis, the two-dimensional gel electrophoresis (2-DE) reference map of a pituitary adenoma tissue proteome is described here. A vertical, two-dimensional (2-D) polyacrylamide gel electrophoresis system and PDQuest image analysis software have been used to provide a high level of between-gel reproducibility and to accurately array each protein expressed in a pituitary adenoma tissue. Mass spectrometry (matrix-assisted laser desorption/ionization-time of flight MALDI-TOF and liquid chromatography-electrospray ionization-quadrupole-ion trap LC-ESI-Q-IT) and protein databases were used to characterize each protein in the 2-D gel. The results demonstrate that a good reproducibility of the 2-D gel pattern was attained. The position deviation of matched spots among four 2-D gels was 1.95 +/- 0.45 mm in the isoelectric focusing direction, and 1.70 +/- 0.53 mm in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis direction. A total of ca. 1000 protein spots were separated by 2-DE, and 135 protein spots that represent 111 proteins were characterized with mass spectrometry (96 spots for MALDI-TOF, 39 spots for LC-ESI-Q-IT). The characterized proteins include pituitary hormones, cellular signals, enzymes, cellular-defense proteins, cell-structure proteins, transport proteins, etc. Those proteins were located in the cytoplasmic, cellular membrane, mitochondrial, endoplasmic reticulum, nuclear, ribonucleosome, extracellular fractions, or were secreted in plasma, etc. Those identified proteins contribute to a functional profile of the pituitary adenoma proteome. These data will be used to expand the proteome database of the human pituitary, which can be accessed in the website http://www.utmem.edu /proteomics.

Functional classification of esterases from leaves of Aspidosperma polyneuron M. Arg. (Apocynaceae)

Polyacrylamide gel electrophoresis system (PAGE) and inhibition tests for biochemical characterization of a- and b-esterases were used to obtain a functional classification of esterases fromAspidosperma polyneuron. The characterization of a- and b-esterases from young leaves of A. polyneuron by the PAGE system showed fourteen esterase isozymes. The differential staining pattern showed that Est-2 isozyme hydrolyzes b-naphthyl acetate; Est-6, Est-7 and Est-8 isozymes hydrolyze a-naphthyl acetate, and Est-1, Est-3, Est-4, Est-5, Est-9, Est-10, Est-11, Est-12, Est-13, and Est-14 isozymes hydrolyze both a- and b-naphthyl acetate. Inhibition pattern of a- and b-esterases showed that Folidol is a more potent inhibitor that Malathion, while Thiamethoxan (an insecticide with organophosphorus-like action) acts as an Est-4 and Est-6 inhibitor and induces the appearance of Est-5 and Est-7 isozymes as more intensely stained bands. Inhibition tests showed that OPC insecticides inhibit or activate plant esterases. Thus, plant esterases may be used as bioindicators to detect the presence and toxicity of residues of topically applied insecticides in agriculture and may be valuable for monitoring pollutants in the environment.

Comparative proteomics: characterization of a two-dimensional gel electrophoresis system to study the effect of aging on mitochondrial proteins

To study the effect of aging and anti-aging strategies on mitochondria, we have characterized a two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system to analyze the profile of mitochondrial proteins. We have optimized the separation of proteins by 2-D PAGE and established the linearity and reproducibility of the system with mitochondria isolated from skeletal muscle of mice. Using total mitochondria protein ranging from 10 to 200 μg, we found that 74% of the proteins resolved by 2-D PAGE had coefficient of determination ( R 2) values greater than 0.8, showing a linear increase in fluorescence with increasing protein concentration. The coefficient of variation (CV) was less than 50% for at least 93% of the 424 spots analyzed for both gel-to-gel variance and animal-to-animal variance. Using mitochondrial protein fractions prepared from skeletal muscle of 18-month-old mice, we show that 10 animals will be sufficient to detect a 100% difference in the 97% (i.e. 505) of the proteins resolved by 2-D PAGE. Thus, 2-D PAGE provides a sensitive and reliable technique for analysis of protein expression in mitochondria.

Real-time dual zymographic analysis of matrix metalloproteinases using fluorescein-isothiocyante-labeled gelatin and Texas-red-labeled casein

Zymography is a widely used technique for identifying the proteolytic activity of enzymes [1–3]. We recently developed a real-time zymographic system for matrix metalloproteinases (MMPs) using fluorescein-isothiocyanate (FITC)-labeled gelatin as a substrate [4]. In this report, we describe an improvement of this technique wherein both Texas-red-labeled casein and FITC-labeled gelatin are incorporated into a single polyacrylamide gel as substrates. The digestion of gelatin and casein by different MMPs is independently observed on the gel using a transilluminator with the appropriate optical filters. With this fluorescent detection system, MMP-3 could be distinguished from MMP-2 and MMP-9 on the same gel and activities could be measured concurrently and in real time. Using this dual detection system, we can save the time and reagent for the experiment and the precise comparison of the digestion bands is accomplished as well. Methods. Collagen was extracted from newborn calf skin by pepsin digestion and purified by sequential salt precipitation [5]. Collagen was labeled with FITC (Dojindo Laboratories, Kumamoto, Japan) in a 0.5M sodium carbonate–sodium hydrogen carbonate buffer (pH 9.5) as previously described [4]. The absorbance at 495 nm for the purified 0.2% FITC–collagen in 1% sodium carbonate solution was 0.939. Calculation from the molecular extinction coefficient of FITC (77,000 at 495 nm) indicated that 1.83 molecules of the FITC bound to each molecule of type I collagen (300 kDa). FITC–collagen was denatured into FITC–gelatin by heating in boiling water when it was used as a substrate for zymography. Commercial casein was from New Zealand Milk Products (Reporoa, New Zealand). A total of 200ml of 0.25% casein was prepared in 0.5M sodium carbonate– sodium hydrogen carbonate buffer, pH 9.0. Ten milligrams of Texas red (Research Organics, OH) was directly added to the casein solution with gentle stirring [6]. The conjugation reaction was carried out at 4 C for 5 h in the dark. We added 6N HCl to the mixture until the pH reached 4.6 (the isoelectric point of casein) to stop the reaction and to precipitate the labeled casein. The precipitate was then dissolved in a solution of NaOH (pH 9.4). The Texas-red-labeled casein was purified by repeating this precipitation–dissolution cycle five times. Next, the purified Texas red–casein was lyophilized. The absorbance at 596 nm for Texas red–casein (0.05%) in the NaOH solution (pH 9.0) was 0.201. Based on the molecular extinction coefficient (84,000 at 596 nm), 0.10 molecules of Texas red were bound to each molecule of casein (27.8 kDa). The sodium dodecyl sulfate–polyacrylamide gel electrophoresis system used was based on the method of Weber and Osborn [7] with several modifications [4]. Zymography was performed under nonreducing conditions in an 8 7 cm (0.75-mm thickness) slab gel containing 12% acrylamide, 0.25mg/ml FITC-labeled gelatin, and 0.5mg/ml Texas-red-labeled casein. A standard stacking gel (5% acrylamide) was used. MMPs in sample buffer (50mM Tris–HCl, pH 6.8, 1% SDS, 0.1% bromphenol blue, and 30% glycerol) were loaded onto a gel without heat-denaturation.The samples were run at a constant voltage (110V) for approximately 2 h with cooling in a water bath (at approximately 10 C). After electrophoresis, the gel was washed four times Analytical Biochemistry 307 (2002) 390–392

Differential esterase expression in leaves of Manihot esculenta Crantz infected with Xanthomonas axonopodis pv. manihotis.

The polyacrylamide gel electrophoresis system (PAGE) and inhibition tests for biochemical characterization of alpha- and beta-esterases were used to obtain a functional classification of esterases in plants and to show a differential expression of esterases as markers of pathogenesis in cassava plants (Manihot esculenta Crantz). The characterization of alpha- and beta-esterases from leaves of M. esculenta by the PAGE system was possible using an extraction solution containing two phenol-complexing agents (PVP-40 and sodium metabisulfite), three antioxidant agents (EDTA, beta-mercaptoethanol, and DTT), and one quinone reducer (ascorbic acid). Fourteen esterase isozymes were detected in young unexpanded leaves of M. esculenta cultivars. The inhibition pattern of alpha- and beta-esterases of M. esculenta showed that Est-9 is an arylesterase, and in the unexpanded leaves of the M. esculenta plants infected with Xanthomonas axonopodis pv. manihotis, the Est-7 beta-esterase showed the characteristic staining of an alpha/beta-esterase. This diffrential expression of Est-7 isozyme in young unexpanded leaves of cassava plants can be used as a marker of pathogenesis after infection with X. axonopodis pv. manihotis.

Genetic diversity of high-molecular-weight glutenin subunit compositions in landraces of hexaploid wheat from Japan

The high-molecular-weight (HMW) glutenin subunit composition of seed storage protein of 174 Japanese hexaploid wheat (Triticum aestivum) landraces have been examined by using sodium dodecyl sulfate polyacrylamide gel electrophoresis system. Twenty four different, major glutenin HMW subunits were identified, and each of the landraces contained three to five subunits and 17 different glutenin subunit patterns were observed for 13 alleles in the landraces. On the basis of HMW glutenin subunits composition, Japanese landraces showed a specific allelic variation, close to Japanese commercial wheats in HMW glutenin subunits, different from those in alien hexaploid wheats. Further, it could be concluded that all common glutenin alleles can be found in the 174 landraces originated from Japan. The variation detected in the glutenin subunits is useful for variety identification, has a bearing on our understanding of hexaploid wheat genetic resource evolution in Japan, and raises questions concerning the nature of this genetic variation.

Separation of Histone Variants and Post‐Translationally Modified Isoforms by Triton/Acetic Acid/Urea Polyacrylamide Gel Electrophoresis

Due to their similarities in size and charge, complete resolution of histones by electrophoresis poses a considerable challenge. The addition of nonionic detergents to the traditional acetic acid/urea (AU) polyacrylamide gel electrophoresis (PAGE) system has afforded an excellent method to separate not only the different modified forms of histones, but also the primary sequence variant subtypes of selected histone species; it is widely used to separate histones with varying levels of acetylation. This unit describes the use of gels containing the nonionic detergent Triton X-100, referred to as Triton/acetic acid/urea (TAU) polyacrylamide gels, for analysis of histones. Also included are support protocols detailing several accessory techniques: assembly of gel plates for the TAU gel, preparation of histones from isolated nuclei in a solubilized form amenable to electrophoresis, and electrophoretic transfer of proteins from these gels to PVDF membranes.

Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis.

Interferon gamma (IFN-gamma) is a potent immunomodulatory lymphokine, secreted by activated T-lymphocytes and NK-cells during the cellular immune response. Actions of IFN-gamma are mediated through binding to the IFN-gamma-receptor, present on most cells, and the subsequent activation of a great magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system. A semiconfluent layer of HeLa cells was grown on tissue culture plates, and changes in protein expression due to 100 U/mL IFN-gamma were investigated at different periods after treatment, using pulse labeling with [35S]methionine/cysteine in combination with 2-D PAGE (IPG). The identity of eight protein spots was elucidated by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), and several variants of the IFN-gamma-inducible tryptophanyl-tRNA synthetase (hWRS) were detected by immunoblotting.

Properties of NADH oxidase fromLactobacillus delbrueckii sspbulgaricus

Lactobacillus delbrueckii spp bulgaricus produces substantial amounts of hydrogen peroxide, yet the enzymology of this process has not been explored. We have partially purified a NADH oxidase (EC 1.6.99.3) from the soluble cell fraction (cytosol) of L delbrueckii ssp bulgaricus, prepared by sonication of the microorganisms in a 0·1 M Tris buffer at pH 7·2. The cytosol was treated with agarose beads containing attached FAD residues, which served to bind the enzyme. The enzyme was eluted with a 0·01 M Tris buffer at pH 7·2 containing 10 μM FAD. This preparation had 0·2 mg ml−1 protein and showed a single major band in a polyacrylamide gel electrophoresis system with a molecular weight of near 30 kDa. Gel filtration chromatography gave a molecular weight of 25 kDa. This enzyme preparation consisted of 4–6 subunits with a molecular weight of 5–6 kDa each, which were held together by FAD. No EDTA-sensitive component was required for activity. The enzyme used β-NADH, but not α-NADH or β-NADPH, to produce stoichiometric amounts of H2O2. The apparent Km with respect to NADH, using production of NAD as a criterion, was 534 μM. The enzyme was active at 4°C and was quite thermostable. Its pH optimum was around pH 7·2. The metabolic function of NADH oxidase in lactobacilli remains obscure, though the H2O2 it generates may have a profound effect on the viability of other organisms. © 1998 Society of Chemical Industry.

Hybridization of a 99Tcm-labelled oligodeoxynucleotide to CAPL RNA.

Oligodeoxynucleotides (ODNs) labelled with an appropriate radionuclide could provide a means to identify serious diseases early on and thereby help initiate treatment at a very early phase. Regardless of important issues like in-vivo stability and membrane passage, the key issue for the oligonucleotide approach is the ability of the radiolabelled ODN to hybridize to the target mRNA. The secondary structure of mRNA does not permit all complementary ODNs to hybridize and a careful selection of the probe with consecutive testing is therefore necessary. This study was initiated to demonstrate hybridization of a 99Tcm-labelled 20-mer ODN to RNA of CAPL (S100A4), a gene reported to be overexpressed in metastatic cancers like breast carcinoma and osteosarcoma. The phosphodiester ODN GX-1 (antisense) and two control sequences (scrambled and random) were conjugated to the bifunctional chelating agent S-benzoyl-mercaptoacetyltriglycine (S-benzoyl-MAG3) and labelled with 99Tcm. The radiolabelled ODNs were purified on a C18 mini-column and characterized on a reverse-phase HPLC system. The radio-chemical purity was > 90% and the product was stable for > 6 h in aqueous medium. The hydrization properties of unlabelled, 32P-labelled and 99Tcm-labelled ODNs to transcribed RNA were studied using polyacrylamide gel electrophoresis (PAGE). Direct hybridization of GX-1 to transcribed RNA was demonstrated. A 50-fold excess of unlabelled ODN over transcribed RNA caused a near to complete consumption of RNA by RNase H activation. In 1:1 proportions of radiolabelled (32P and 99Tcm) ODNs to RNA, only radiolabelled GX-1 was found to hybridize to RNA in a PAGE system. The radiolabelled control ODNs did not show signs of hybridization. This study demonstrates that 3'-99Tcm-labelling of ODNs does not interfere with the hybridization properties of the ODNs in solution, making 99Tcm-labelling an attractive procedure for the future development of antisense technology in imaging.

Isolation of ultrapure supercoiled plasmid-DNA using preparative electrophoresis.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) system for the isolation of high-purity supercoiled plasmid-DNA is described. This method should prove suitable for the isolation of large DNA molecules, either plasmid or linear DNA, that is required for the production of transgenic animals, for instance. The efficiency of the method is illustrated by the isolation of the gene for the green fluorescent protein, cloned into a mammalian expression vector and used for transfection of eukaryotic cells.

Synthesis and two-dimensional electrophoretic analysis of mixed populations of circular and linear RNAs

Spontaneous cleavage of the less abundant form of tobacco ringspot virus satellite RNA is readily reversible. Capitalizing on earlier observations by Feldstein and Bruening that small 'mini-monomer' RNAs derived from this molecule and containing little more than covalently attached ribozyme and substrate cleavage products are able to efficiently circularize, we have constructed a series of self-circularizing RNAs of precisely known size. Mixtures of linear and circular RNAs synthesized in vitro and containing 225-1132 nt could be completely resolved using a novel two-dimensional denaturing polyacrylamide gel electrophoresis system. Similar analyses of a complex mixture of coconut cadang-cadang viroid RNAs revealed the presence of relatively large amounts of a previously undescribed 'fast-slow' heterodimeric RNA species in infected palms. Only a single DNA template is required to prepare each pair of circular and linear RNA markers.

Purification, Identification, and Partial Characterization of a Barley Protein that Inhibits Green Malt Endoproteinases

Endoproteinases control the rate of hydrolysis of storage proteins during barley germination and are thus critically important to the malting process. We have shown that endoproteinases comprising all four proteinase classes are present in green malt, with the cysteine proteinases probably being most important for hydrolyzing storage proteins during malting. Compounds from both barley and malt inhibit some of these cysteine proteinases. This article reports the purification and characterization of a 10-kDa barley protein, purified from both seed and beer extracts, that specifically inhibits green malt cysteine endoproteinases. Amino acid composition, matrix-assisted laser desorption/ionization mass spectrometric and N-terminal amino acid sequence data indicated that the inhibitor is identical to barley lipid transfer protein 1 (probable amylase/proteinase inhibitor, PAPI), a nonspecific lipid-transfer protein. The protein did not inhibit the activities of either papain or subtilisin but did suppress the activities of many of the green malt cysteine endoproteinase activities that are separated on a two-dimensional isoelectric focusing and polyacrylamide gel electrophoresis system. Some serine proteinases were also partially inhibited. The purified inhibitor totally inhibited the activity of a purified 31-kDa cysteine endoproteinase from green malt. In the absence of inhibitor, the 31-kDa enzyme rapidly hydrolyzed barley storage proteins. LTP1-PAPI may well play an important role in controlling protein hydrolysis during malting.

Allele frequency distributions of D1S80 in the Polish population

The polymorphism of the D1S80 locus has been analyzed in a population sample of 208 unrelated individuals in the Southeast Poland and 103 mother/child pairs. PCR amplified alleles were separated by a vertical discontinuous polyacrylamide gel electrophoresis system. Nineteen different alleles and 52 phenotypes could be distinguished. The alleles 18 ( f = 0.267) and 24 ( f = 0.300) were most common in Poland. D1S80 genotype frequencies of Poland population do not deviate from Hardy-Weinberg equilibrium. All mother/child pairs shared at least one D1S80 allele.

Electrophoretic Separation of βA4 Peptides (1–40) and (1–42)

Different sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) systems designed for the separation of peptides were compared for their usefulness in separating synthetic β-amyloid peptides βA4 (1–40) and βA4 (1–42). Clear resolution was achieved by addition of 8Murea to the separation gel and use of a multiphasic buffer system employing bicine and sulfate as trailing and leading ions, respectively (bicine/Tris/urea gels). Under these conditions, the longer peptide migrated faster than the one ending at amino acid 40. The usefulness of this SDS–PAGE system for the analysis of βA4-related peptides generated during cellular metabolism was demonstrated by immunoprecipitation and electrophoretic separation of radiolabeled peptides secreted by cells transfected with amyloid precursor protein cDNAs.

Identification and evaluation of the role of endogenous tyrosine kinases in azoxymethane induction of proliferative processes in the colonic mucosa of rats

Although tyrosine kinases (Tyr-k) are known to play a role in regulating proliferation of normal, preneoplastic and neoplastic cells, little is known about the identity of different species of Tyr-k involved in this process. Utilizing a non-denaturing polyacrylamide gel electrophoresis system, in which the separated proteins from tissue extracts are assayed directly for Tyr-k, we attempted to identify the species of Tyr-k that may be involved in azoxymethane (AOM) induction of colonic mucosal ornithine decar☐ylase (ODC) activity, an enzyme whose activity is known to rise in rapidly proliferating cells. We have observed that 5 days after a single injection of the colonic carcinogen AOM (20 mg/kg body wt.) to 3–4-month old rats, a significant 230% rise in colonic mucosal proliferative activity (as evidenced by 5-bromo-2′-deoxyuridine (BrdU) immunoreactivity) was also accompanied by a 550% increase in ODC activity. This was also associated with a marked rise (140–240%) in the relative activity of Tyr-k of three mucosal proteins with M r of 165, 145 and 125 kDa. Since the molecular mass of one of the Tyr-k (165 kDa) corresponded to that of EGF-receptor (EGF-R), this led us to examine the role of EGF-R Tyr-k in AOM induction of colonic mucosal ODC. We observed that a 320% increase in mucosal ODC activity, 5 days after AOM injection, was accompanied by over 200% rise in Tyr-k activity of EGF-R. Daily injection of tyrphostin (300 μg/kg body wt.), a Tyr-k inhibitor with a higher specificity for EGF-R Tyr-k, significantly attenuated AOM-induced stimulation of both ODC and Tyr-k activity of EGF-R. Administration of AOM also stimulated the rate of synthesis and secretion of TGF-α in isolated colonocytes. In addition, the levels of TGF-α and its mRNA in the colonic mucosa were also found to be 100% and 250% higher, respectively, in AOM-treated rats when compared with the controls. We suggest that (a) activation of intrinsic Tyr-k of EGF-R is an important event in AOM induction of colonic mucosal proliferative processes, and (b) this activation is thought to be mediated by TGF-α through an autocrine mechanism.

An Improved SDS–Polyacrylamide Gel Electrophoresis for Resolution of Peptides in the Range Of 3.5-200kDa

Resolution of a wide range of polypeptides, 3.5-200kDa, on a single low acrylamide and cross linkage gel of 7.7% T, 2.6% C is described here. Laemmli's (4) original discontinuous SDS polyacrylamide gel electrophoresis (SDS-PAGE) system is modified by increasing the ionic strength of both stacking and resolving gels, and replacing the usual glycine buffer with a tricine cathode buffer as described by Schagger and von Jagow (7). This system offers the advantage of a wide range of protein fractionation, with sufficient band resolution, on a single, low acrylamide concentration and cross linkage gel. Moreover, increased gel ionic concentration allows higher protein and salt load, and renders this system suitable for preparative work.