Biochemical Identification of the Two Races of Radopholus similis by Polyacrylamide Gel Electrophoresis

Analysis of proteins of the banana and citrus race of Radopholus similis was carried out by several different types of polyacrylamide gel electrophoresis. These included standard slab gel, SDS slab gel, gradient slab gel, and two-ditnensional slab gel electrophoresis. A major band difference was detected between the two races by slab gel electrophoresis. However, several other poorly resolved but consistent hands of high molecular weight proteins near the gel origin also were considered as diagnostic. Resolution of protein bands was greatly improved by SDS and gradient slab gel electrophoresis, but no differences could be detected among the proteins resolved between the two rares with these techniques. Two-dimensional gels revealed a large number of proteins, but background staining obscured them hindering interpretation. When nematode races were reared on three different host plants, no differences in protein patterns were detected between them, indicating host preferences does not play a role in determining the types proteins occurring in these nematodes.     

Two-dimensional electrophoresis of plasma proteins without denaturing agents.

A technique of two-dimensional polyacrylamide gel electrophoresis for the separation of plasma proteins is described. Human plasma proteins were separated by isoelectric focusing followed by electrophoresis in a 4 to 21% linear gradient gel slab. No denaturing agent was used throughout the procedure, so that the analysis of native proteins is possible. Two-dimensional patterns obtained from normal human plasma samples were recorded as "staining density maps," which are similar to contour line maps, and more than 230 protein spots were counted reproducibly on each "staining density map." This technique permits the simultaneous estimation of pI's and approximate molecular weights of native proteins on the slab gel. Applications of this technique to an IgA myeloma plasma sample and a porcine serum sample are described.     

Hydroxylamine cleavage of proteins in polyacrylamide gels

A modification of the hydroxylamine cleavage of proteins is presented in which proteins were cleaved while immobilized in the matrix of a polyacrylamide gel. The reaction under these conditions retains its high specificity for Asn-Gly bonds and has the advantage that the gel matrix, acting as a carrier, facilitates simultaneous treatment of many samples, and contributes to a high recovery efficiency (60-90%) of the cleavage products. The cleavage is performed with individual protein bands excised from dried slab gels after detection by staining, autoradiography, or fluorography. The procedure can be easily combined with other techniques to further characterize the cleavage fragments. Also a two-dimensional version of the cleavage method was developed, which allows rapid recognition of interrelationships between proteins in a complicated mixture. The versatility of the procedure is demonstrated in a number of applications. Highly related strains of murine leukemia viruses were easily distinguished from one another by the unique cleavage patterns of their gag- and env-precursor polypeptides. Comparing the env-precursor gPr82env synthesized in the presence or absence of tunicamycin with its cell-free synthesized counterpart, revealed the presence of an amino-terminal signal sequence. Cleavage patterns of pro-opiomelanocortin (POMC) from three different species revealed a high degree of homology between rat and mouse POMC, whereas Xenopus POMC was very different. Regions to which carbohydrates are attached could be identified by comparing glycosylated and unglycosylated forms of POMC. Combining the hydroxylamine cleavage procedure with immunological characterization of the fragments showed a small but significant difference between the amino-terminal sequences of the recombinant transforming protein P120 of Abelson murine leukemia virus and of its parent molecule Pr65gag of Moloney murine leukemia virus.     

Determination of collagen band positions on polyacrylamide gel without staining

Protein band positions on a slab polyacrylamide gel were determined without staining, by blotting the bands onto a nitrocellulose membrane which was then stained to visualize the electrophoretic pattern. The unfixed, unstained bands were cut from the gel according to the copy pattern placed underneath the slab gel. Collagen alpha 1 and alpha 2 chains were separately extracted from the gel using this procedure.     

Purification of protein from a crude mixture through SDS-PAGE transfer method

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) transfer method was used for purification and enrichment of the protein from crude sample. Coomassie bluc/ZnSO4 stained protein band(s) containing intact polyacrylamide gel were loaded on to another polyacrylamide gel either alone or as pooled gel bands. Two/three bands were combined together and arranged tightly over one another, sealed with stacking gel and ran in another gel, which was quite useful for enrichment and purification of a particular protein from a complex mixture. Recovery of protein by gel transfer method was found to be 70% in case of ZnSO4 staining, whereas around 30% recovery was possible, following Coomassie blue staining. The method described here for purification of protein(s) from a complex mixture, following gel transfer procedure could be useful for further characterization of the desired protein.     

Characterization of sucrose-hydrolyzing enzymes of Zymomonas mobilis

Extracellular proteins of Zymomonas mobilis were analyzed by two-dimensional gel electrophoresis and protein maps drawn up. One of these proteins showed sucrose-hydrolyzing activity, as indicated by activity staining after polyacrylamide gel electrophoresis. It was purified from the extracellular extract of a glucose fermentation by polyacrylamide gel electrophoresis, using a two-step procedure. The molecular mass of the protein was 46 kDa and its isoelectric point 5.0. A rabbit antiserum was raised against this protein. As shown by immunoblotting, the same protein was present in extracellular extracts obtained from glucose, fructose and sucrose fermentations. A cross-reaction was also detected by immunoblotting, with a cellular protein of molecular mass 46 kDa present on the three carbon sources studied. However, activity staining was unsuccessful on gels after electrophoresis of these cellular extracts. The extracellular protein extract obtained from a fermentation run on glucose contained another sucrose-hydrolyzing protein of molecular mass 51 kDa and with an isoelectric point of 4.8. This protein was absent in fructose and sucrose fermentations but showed a positive reaction with the antiserum raised against the 46 kDa extracellular protein. Partially purified sucrose-hydrolyzing proteins also catalyzed transfructosylation reactions, suggesting that they could be of the levansucrase type.     

Quantitation of proteins on Coomassie blue-stained polyacrylamide gels based on spectrophotometric determination of electroeluted dye

A procedure for quantitating proteins on Coomassie blue-stained polyacrylamide gels is presented. The method is based on the observations that the dye is rapidly eluted electrophoretically from stained protein bands or spots in the presence of sodium dodecyl sulfate, and that the eluted dye is nondialyzable. Protein may therefore be assayed indirectly by measuring the dye in electroeluents spectrophotometrically. Moreover, the stain elutes more rapidly than the protein, allowing separate recovery of the protein for further analysis. The assay is independent of band or spot size, and does not involve physical disruption of the gel piece or any chemical treatment harsher than the staining process itself. The technique has been applied to the contractile proteins myosin, actin, and commercially obtained standards resolved by one-dimensional electrophoresis, and to proteins in nuclear extracts of HeLa cells on two-dimensional gels.     

Peptide mapping of protein bands from polyacrylamide gel electrophoresis by chemical cleavage in gel pleces and re-electrophoresis

A simple method has been developed for peptide mapping of protein bands obtained by polyacrylamide gel electrophoresis. The procedure is based on selective acid hydrolysis of aspartyl-prolyl bonds which occur in proteins with an average frequency of 1 per 400 amino acid residues. A gel piece containing the protein to be analyzed is soaked with 75% formie acid. For the subsequent incubation at 37°C for 24 h the gel piece is immersed in liquid paraffin. After removal of formic acid by lyophilization the gel piece is rehydrated in buffer and placed into the sample well of a second polyacrylamide gel on which the generated peptides are electrophoretically separated.     

Nongradient blue native gel analysis of serum proteins and in-gel detection of serum esterase activities

The objective of the present study was to analyze serum protein complexes and detect serum esterase activities using nongradient blue native polyacrylamide gel electrophoresis (BN-PAGE). For analysis of potential protein complexes, serum from rat was used. Results demonstrate that a total of 8 gel bands could be clearly distinguished after Coomassie blue staining, and serum albumin could be isolated nearly as a pure protein. Moreover, proteins in these bands were identified by electrospray mass spectrometry and low-energy collision induced dissociation (CID)-MS/MS peptide sequencing and the existence of serum dihydrolipoamide dehydrogenase (DLDH) was confirmed. For studies of in-gel detection of esterase activities, serum from rat, mouse, and human was used. In-gel staining of esterase activity was achieved by the use of either α-naphthylacetate or β-naphthylacetate in the presence of Fast blue BB salt. There were three bands exhibiting esterase activities in the serum of both rat and mouse. In contrast, there was only one band showing esterase activity staining in the human serum. When serum samples were treated with varying concentrations of urea, esterase activity staining was abolished for all the bands except the one containing esterase 1 (Es1) protein that is known to be a single polypeptide enzyme, indicating that majority of these esterases were protein complexes or multimeric proteins. We also identified the human serum esterase as butyrylcholinesterase following isolation and partial purification using ammonium sulfate fractioning and ion exchange column chromatographies. Where applicable, demonstrations of the gel-based method for measuring serum esterase activities under physiological or pathophysiological conditions were illustrated. Results of the present study demonstrate that nongradient BN-PAGE can serve as a feasible analytical tool for proteomic and enzymatic analysis of serum proteins.     

Enzymatic, isoelectric, and molecular-weight characterization of water-soluble maize-pollen proteins

Maize (Zea mays L. cv. Kansas Drought Synthetic) pollen proteins were fractionated via a continuous tandem cascade ultrafiltration system into three molecular-weight classes. Proteins from the whole extract and each fraction were further separated by polyacrylamide gel electrofocusing in the pH 5 to 9 range and characterized by their enzymatic activity and isoelectric point. Twenty-one protein bands were detected with Fast Green FCF staining. Esterase, peroxidase, leucine aminopeptidase, catalase, and amylase activities were located in 16 bands. Five bands remained unclassified. All the enzymes assayed, except amylase, were polymorphic. The extractions, fractionations and zymograms were reproducible and indicated that several protein bands contained at least two different enzymes with very similar molecular weight class and isoelectric point for each enzyme and protein band     

A new enzymatic staining method for the detection of radish beta-fructosidase in gel electrophoresis

Optimum conditions for β-fructosidase detection in polyacrylamide and agarose gels are defined from comparison of zymograms obtained with two staining methods including an original one. Under all conditions tested in the present study detection has been improved with the new staining procedure. The new staining medium developed here uses two intermediary enzymes: glucose oxidase and peroxidase, 3.3′-diaminobenzidine as final acceptor, and sucrose as substrate for β-fructosidase. β-Fructosidase zymogram is obtained either by gel immersion in this staining solution, or when highly crosslinked polyacrylamide separating gels are used, by overlaying the slab within a thin buffered agarose gel containing staining chemicals (“sandwich technique”). Examples are presented to illustrate the general principles involved and indicate the conditions necessary for optimal development of this precise and specific technique.     

Detection of cellulase activity in polyacrylamide gels using Congo red-stained agar replicas

Bands that have cellulolytic activity are visualized after polyacrylamide gel electrophoresis by laying the slab gel on top of a thin sheet of 2% agar containing 0.1% carboxymethylcellulose. After a suitable incubation time, zones of carboxymethylcellulose hydrolysis are revealed by staining the agar replica with Congo red.     

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

Separation of apolipoprotein B species by agarose-acrylamide gel electrophoresis

Human apolipoprotein (apo) B has been recognized to exist in two different forms designated apoB-100 and apoB-48. The two apoB forms are usually separated by NaDodSO4 gel electrophoresis with a low percentage polyacrylamide gel in a tube gel apparatus. However, the matrix of this low percentage gel is relatively weak, and one can separate the two forms of apoB in a slab gel apparatus only if one utilizes a gradient polyacrylamide gel or a higher percentage polyacrylamide gel which results in a poorer separation of the protein bands. We have developed an agarose-acrylamide gel electrophoretic method to separate the two major apoB forms. The gel is a mixture of 0.5% agarose and 2% acrylamide. The agarose-acrylamide method is fast, has the advantage of being able to be used on an analytical or preparative scale in a vertical slab gel apparatus, and the gel is of sufficient strength to be used in immunoblotting and/or radioautography.     

Oxidative metabolism of dopamine: a colour reaction from human midbrain analysed by mass spectrometry

In order to identify the protein responsible for a dopamine peroxidizing activity, previously described in human normal and parkinsonian substantia nigra by our group, we developed non-denaturing polyacrylamide gel electrophoresis conditions, mimicking the characteristic colour in vitro reaction, resulting from cyclic oxidation of dopamine (DA). After separating protein mixtures from human normal midbrain homogenates on two sets of identical native gels, one gel set was subjected to specific activity staining by using DA and hydrogen peroxide. An activity red/orange band appeared in midbrain tissue lanes, similarly to the lane where commercial horseradish peroxidase (HRP) was present as control of peroxidative activity. The second set of gels, stained with Coomassie Blue, showed other, not enzymatically active protein bands. Mass spectrometry analysis of the bands containing the activity and the corresponding Coomassie Blue bands revealed the presence of proteins that may play a role in neurodegenerative disease, highlighting a possible functional link among dopamine/dopaminochrome redox cycle and protein metabolism.     

Visible fluorescent detection of proteins in polyacrylamide gels without staining

2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization. This study shows that placing TCE in the gel before electrophoresis can eliminate the staining step. The gel is removed from the electrophoresis apparatus and placed on a transilluminator and then the protein bands develop their fluorescence in less than 5min. In addition to being rapid this visualization method provides detection of 0.2microg of typical globular proteins, which for some proteins is slightly more sensitive than the standard Coomassie brilliant blue (CBB) method. Integral membrane proteins, which do not stain well with CBB, are visualized well with the TCE in-gel method. After TCE in-gel visualization the same gel can then be CBB stained, allowing for complementary detection of proteins. In addition, visualization with TCE in the gel is compatible with two-dimensional PAGE, native PAGE, Western blotting, and autoradiography.     

The Immunological Identification of Brain Proteins on Cellulose Nitrate in Human Demyelinating Disease

Abstract: An immunological technique has been employed to identify proteins, separated in polyacrylamide gels, which show changes in brain samples from cases of multiple sclerosis and subacute sclerosing panencephalitis. Sodium dodecylsulphate-treated proteins in particulate and soluble fractions were separated in polyacrylamide slab gels, transferred electrophoretically onto cellulose nitrate sheets, incubated with specific antisera and visualized by an immunoperoxidase method. Protein bands showing changes were identified using antisera raised against the myelin basic and Wolfgram proteins, the neurofilament triplet proteins, tubulin and glial fibrillary acidic protein. In addition to the loss of myelin proteins, decreases in the neurofilament proteins and in tubulin were seen in both multiple sclerosis and subacute sclerosing panencephalitis samples. The distribution of glial fibrillary acidic protein polypeptides in the particulate and soluble fractions of plaque samples appeared to vary according to the degree of fibrosis. Changes in the levels of the myelin-associated glycoprotein, the lower molecular weight component of the Wolfgram protein, albumin and immunoglobulin G, none of which were visualized by protein staining, were also seen. This immunological technique has allowed a closer examination of changes occurring in brain protein spectra in multiple sclerosis and subacute sclerosing panencephalitis.     

Isolation of outer membrane proteins of Escherichia coli and their characterization on polyacrylamide gel

Proteins from the outer membrane of Escherichia coli were studied on a ureadodecyl sulfate polyacrylamide gel by electrophoresis. A polyacrylamide gel containing sodium dodecyl sulfate and urea gave an excellent resolution of outer membrane proteins. Seventeen protein bands were reproducibly observed on a gel. By use of Sephadex G-200, DEAE-cellulose and polyacrylamide gel, eight proteins were purified to near homogeneity. Five of them were found to be heat-modifiable proteins. The behavior of these purified proteins was studied on a polyacrylamide gel under three different electrophoretic conditions, which had been used for the analysis of cell envelope proteins. Thus correspondence was made between these purified proteins and envelope proteins reported by other investigators.     

SDS聚丙烯酰胺凝胶电泳快速染色新方法的研究

通过几种金融盐溶液对SDS聚丙烯酰胺凝胶电泳染色的实验表明,0.25mol/L的CaCl2和MgCl2溶液能够对蛋白质进行有效的染色,经这2种溶液染色的蛋白质都能够从凝胶中洗脱回收。尤其是CaCl2法灵敏度更高,而且蛋白质条带形成之后也十分稳定,所以在运用制备电泳纯化蛋白质时这种新的染色方法较适用。     

Purification and characterization of 8 of the pathogenesis-related proteins in tobacco leaves reacting hypersensitively to tobacco mosaic virus

Summary Leaves of tobacco plants (Nicotiana tabacum cv. Samsun NN) which are reacting hypersensitively to infection with tobacco mosaic virus contain 10 major pathogenesis-related (PR) proteins which are absent, or present in small amounts in uninfected leaves. We describe here a preparative procedure of purification of the tobacco PR-proteins which involves a combination of conventional and high-performance liquid chromatography. The separation and isolation of the proteins were based on differences in net charge at different pH values, in isoelectric point and in apparent molecular weight. This procedure led to the purification to homogeneity of 8 PR-proteins, as shown by polyacrylamide slab gel electrophoresis (PAGE) of the purified proteins under denaturing and non-denaturing conditions. These were the 3 well-known proteins PR-1a,-1b and-1c, and 5 other major PR-proteins, called PR-2,-N,-O,-P and-Q, according to the nomenclature of Van Loon (39). None of the purified PR-proteins gave a positive Schiff reaction for carbohydrate content. Molecular weight determinations from gel permeation chromatography and from sodium dodecyl sulphate (SDS)-PAGE indicated that all 8 PR-proteins were monomers and that three groups could be distinguished among them. The first group is the PR-1 group containing PR-1a,-1b and-1c (12000 MW), the second consists of PR-P and PR-Q (14000 MW) and the third of PR-2, PR-N and PR-O (25000 MW). In the PR-1 group, PR-1a can be distinguished clearly from the two other members on denaturing slab gels containing both SDS and urea.