Detection by the use of silver ions of additional protein zones in gradient polyacrylamide gels stained with Coomassie

A simple method of revealing the additional zones of proteins in gradient polyacrylamide gels, preliminary dyed Coomassie by means of silver ions is described. The dyeing of Coomassie allows to avoid the time-consuming stages of preliminary treatment of gels as well to reveal more sensitive zones in gels. On the second stage of dyeing silver minor zones appear there which were not seen while Coomassie was dyed. The suggested method preserves high sensitivity characteristic of the methods of gel dyeing with silver.     

The instantaneous monitoring of polyacrylamide gels during electrophoresis.

The advantages of being able to see protein zones in a gel during electrophoresis (and hence before staining) are pointed out, and a method is described which depends on local increments of refractive index in these zones. The use of local increments of refractive index in polyacrylamide gels for measuring protein concentrations in zones during electrophoresis is briefly considered; it is found that such increments are greater than would be expected from the amount of protein when sodium dodecyl sulphate is present. The enhancement depends on conditions and time of running. This makes quantitative estimates difficult, but the sensitivity of detection of protein zones by observations based on refractive-index changes is greatly increased by this property of sodium dodecyl sulphate. Methods are described for making optically uniform gels (both with uniform and with graded concentrations of polyacrylamide), necessary for observation of small changes in refractive index. A simple dark-field system of observation is described. Examples are given showing protein samples observed with the system during electrophoresis and compared with the same gel stained with Coomassie Blue after completion of the run. Under optimal conditions the optical method is comparable in sensitivity with staining. With the proteins of lower mol.wt. (approx. 15000), the optical method is not so sensitive, becoming less sensitive with longer running time. This loss of sensitivity is greatly decreased by using more concentrated polyacrylamide gels, and graded gels are therefore more suitable for optical observation than are uniform gels. The observation of protein zones during electrophoresis adds nothing to the time needed for making a stained gel and gives much information long before it can be obtained from the stained gel.     

Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels

Current methods for quantitatively comparing proteomes (protein profiling) have inadequate resolution and dynamic range for complex proteomes such as those from mammalian cells or tissues. More extensive profiling of complex proteomes would be obtained if the proteomes could be reproducibly divided into a moderate number of well-separated pools. But the utility of any prefractionation is dependent upon the resolution obtained because extensive cross contamination of many proteins among different pools would make quantitative comparisons impractical. The current study used a recently developed microscale solution isoelectrofocusing (musol-IEF) method to separate human breast cancer cell extracts into seven well-resolved pools. High resolution fractionation could be achieved in a series of small volume tandem chambers separated by thin acrylamide partitions containing covalently bound immobilines that establish discrete pH zones to separate proteins based upon their pIs. In contrast to analytical 2-D gels, this prefractionation method was capable of separating very large proteins (up to about 500 kDa) that could be subsequently profiled and quantitated using large-pore 1-D SDS gels. The pH 4.5-6.5 region was divided into four 0.5 pH unit ranges because this region had the greatest number of proteins. By using very narrow pH range fractions, sample amounts applied to narrow pH range 2-D gels could be increased to detect lower abundance proteins. Although 1.0 pH range 2-D gels were used in these experiments, further protein resolution should be feasible by using 2-D gels with pH ranges that are only slightly wider than the pH ranges of the musol-IEF fractions. By combining musol-IEF prefractionation with subsequent large pore 1-D SDS-PAGE (>100 kDa) and narrow range 2-D gels (<100 kDa), large proteins can be reliably quantitated, many more proteins can be resolved, and lower abundance proteins can be detected.     

A specific peroxidase-coupled activity stain for diamine oxidases.

A specific peroxidase-coupled activity staining method for diamine oxidase (DAO) was developed. Diaminobenzidine was found to inhibit DAO and to give rise to unspecific staining. Among several other reagents 4-Cl-1-naphthol was found to be most suitable. Using specific activity staining DAO could be visualized in polyacrylamide gels as a high-molecular-weight complex, which could be dissociated by Tween 20 but not by NP-40, Triton X-100, or Chaps.     

Genetic Variation of the Equine Serum Protease Inhibitor System Pi (Pr) Characterized By an Enzyme Binding Staining Technique After Starch Gel Electrophoresis

A modification of Uriel & Berges (1968) staining technique has been developed for starch gels. This method, which makes use of the Pi proteins ability to bind trypsin and chymotrypsin, allows for the recognition of the Pi zones which migrate into slower positions than originally described by Braend (1970). The Pi zones appear as white bands against a lilac background. Serum samples from 18 sire families selected according to the Pi type of the sires have been studied. Ten families were Norwegian Trotter, 8 were Warm-blood Trotter (Standardbred). In each family 12 dam-offspring pairs were examined. In trypsin-treated gels the white zones usually correspond to those previously recognized by protein-staining. In addition, the products of the PiG, Pi1, PiL and PiW alleles each had 1 distinct slow band, but in different positions. The products of the PiN and PiU alleles lacked slow zones. The Pis and PiT alleles differed with respect to the positions of their slow bands. A new allele PiT1 was identified. This has a slow band in a different position from that of the PiL allele. An allele indistinguishable from PiZ was recognized in Norwegian Trotter in which also a new alle Je temporarily called PiY could be demonstrated. In chymotrypsin-treated gels the zone patterns of some of the allele products differed from those seen after trypsin-treatment.     

Reexamining the egg-box model in calcium-alginate gels with X-ray diffraction

The structure of the Ca--alginate junction zones was investigated with X-ray scattering on gels prepared with different methods. Fiber diffraction reveals the popular egg-box model may not be the only possible structure for the junction zones. The (001) reflection, which should be extinguished due to 2/1 helical conformation in the egg-box model, was observed. This was further confirmed by the measurements on Ca--alginate gel beads prepared at different pH where large pieces were formed through a relatively slow process, which leads to a higher crystallinity and a more perfect ordering. The results suggest a 3/1 helical conformation is more proper for Ca--alginate gels formed slowly. This does not exclude the possibility for the 2/1 helical conformation in fast gelatinized Ca--alginate in which the 2/1 helix is a metastable form. Comparing the X-ray scattering results of the fresh, dehydrated, and rehydrated gels, a reversible aggregation of junction zones is found during dehydration and rehydration. The different stabilities of initial bonds and bonds formed during drying are speculated to be the contribution of MG block or short G blocks in the junction zones.     

Conformations and interactions of pectins: II. Models for junction zones in pectinic acid and calcium pectate gels

Pectinic acid and calcium pectate gels condensed into uniaxially oriented fibers have been studied by X-ray diffraction. Although the diffraction patterns correspond to systems of only limited order, they show that both systems conserve the 1.3 nm axial period and 0.43 nm pseudo-period observed in sodium pectate. Pectinic acid further resembles sodium pectate in packing isometrically in a hexagonal net of side 0.84 nm. On the other hand, calcium pectate fibers contain the 1.2 nm lateral spacing observed in pectic acid. Speculative models for pectinic acid and calcium pectate have been developed. The former structure could be stabilized by hydrophobic binding from columns of methyl groups as well as by specific intermolecular hydrogen bonds. In the latter, the main interactions between pairs of chains could be bridges formed by calcium ions, which incorporate into their co-ordination shells two polyanion oxygen atoms from one chain and three from another. These model-building studies provide plausible visualizations of two different kinds of junction zones that may exist in pectic gels.     

High-Resolution Detection of PCR Products from a Microsatellite Marker Using a Nonradioisotopic Technique

We report a safe, rapid, and economical method for polymerase chain reaction (PCR)-based genotype analysis using a microsatellite marker specific for the human chromosome 18 locus, D18S53. This method does not involve radioisotopes and makes use of ethidium bromide fluorescence to detect PCR products. Our method enables direct analysis and easy detection of PCR products on nondenaturing polyacrylamide gels. The genotyping using this method can be scaled up to 100 samples at one time by adding a step of “double loading” of samples in a single sequencing size gel. We could resolve PCR products and DNA fragments, differing in size by only 2 bp, in the range of 150–200 bp by a 7% nondenaturing polyacrylamide gel. This technique can be applied for population-based genomic screening and linkage analysis.     

Selective elution of zones from preparative isoelectric focusing gels by ampholytes or buffers.

Protein zones formed by isoelectric focusing on polyacrylamide gels (IFPA) can be eluted without mechanical disruption of the gels. Specific elution is achieved by replacement of the original anolyte, a strong acid, with an ampholyte of a pI higher than that of the protein which is to be eluted. Alternatively, the anolyte may be a buffer of a pH higher than the pI of the focused protein zone. A rudimentary apparatus and procedures for the application of this method of zone elution are described but are not as yet sufficiently developed to provide a ready-to-use preparative IFPA procedure.     

A procedure to increase the sensitivity of staining by Coomassie brilliant blue G250-perchloric acid solution.

The intensity of staining of protein zones in polyacrylamide gels by Coomassie brilliant blue G250 in perchloric acid solution was increased by a factor of 3 when a wash of 5% acetic acid followed staining. Concentrations as low as 5 ng of human serum albumin could be detected in the gels.     

The resolution of membrane proteins based upon size,charge, and hydrophobicity

Currently available systems for resolving membrane proteins are based only on size and charge differences. Recently, it has been shown that Triton-urea-acetic acid gels which separate proteins on the basis of charge, size and hydrophobicity are capable of resolving proteins differing only by the substitution of a single neutral amino acid. We have applied this new method to the resolution of bacterial envelope proteins. Conditions for optimal resolution of different bacterial envelope proteins were determined by electrophoresis through transverse urea and Triton X-100 gradient gels. We have also correlated the components resolved in this system with those resolved by classical sodium dodecyl sulfate-gel electrophoresis by using two-dimensional slab gels combining the two systems. Furthermore, envelope protein fractions from different species and strains of bacteria were compared to identify specific proteins. This system appears to be a promising method for investigating envelope proteins which are due to missense mutations.     

Differential regulation of beta1 integrins by chemoattractants regulates neutrophil migration through fibrin

Chemoattractants differ in their capacity to stimulate neutrophils to adhere to and to migrate through matrices containing fibrin. Formyl methionyl leucyl phenylalanine (fMLP) stimulates neutrophils to adhere closely to, but not to migrate into, fibrin gels. Leukotriene B4 (LTB4) stimulates neutrophils to adhere loosely to and to migrate through fibrin gels. We report that alpha5beta1 integrins regulate the different migratory behaviors on fibrin gels of neutrophils in response to these chemoattractants. fMLP, but not LTB4, activated neutrophil beta1 integrins, as measured by binding of mAb 15/7 to an activation epitope on the beta1 integrins. Antibodies or peptides that block alpha5beta1 integrins prevented fMLP-stimulated neutrophils from forming zones of close apposition on fibrin and reversed fMLP's inhibitory effect on neutrophil chemotaxis through fibrin. In contrast, neither peptides nor antibodies that block beta1 integrins affected the capacity of LTB4-stimulated neutrophils to form zones of loose apposition or to migrate through fibrin gels. These results suggest that chemoattractants generate at least two different messages that direct neutrophils, and perhaps other leukocytes, to accumulate at specific anatomic sites: a general message that induces neutrophils to crawl and a specific message that prepares neutrophils to stop when they contact appropriate matrix proteins for activated beta1 integrins.     

Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenases in mouse liver

Summary The precise histochemical localization and quantification of the activity of soluble dehydrogenases in unfixed cryostat sections requires the use of tissue protectants. In this study, two protectants, polyvinyl alcohol (PVA) and agarose gel, were compared for assaying the activity of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) in normal female mouse liver. Quantification of enzyme activity was determined cytophotometrically in periportal (PP), pericentral (PC) and midzonal (MZ) areas. No coloured reaction product was present in PVA media after the incubation period. In contrast, the agarose gels appeared to be highly coloured after incubation. As a consequence, sections incubated with gel media were less intensely stained than those incubated in PVA-containing media. The specific G6PDH reaction (test minus control) yielded approximately 75% less formazan in sections incubated by the agarose gel method than with the PVA method. Further, the amount of formazan deposits attributable to G6PDH activity was highest in the midzonal and pericentral zones of the liver lobule with PVA media, and Kupffer cells could be discriminated easily because of their high G6PDH activity. Significant zonal differences or Kupffer cells could not be observed when agarose gel films were used for the detection of G6PDH activity. The LDH localization patterns appeared to be more uniform after incubation with both methods: no significant differences in specific test minus control reactions were seen between PP, PC and MZ. However, less formazan production (33%) was detected in sections incubated with agarose gels when compared with those incubated with PVA media. These results clearly show that the gel method is not suitable for the valid demonstration of activity of (partially) soluble enzymes. Furthermore, our results confirm that a greater proportion of G6PDH than of LDH is present in a soluble form in liver cells.     

Multilocus enzyme analysis in aerobic and anaerobic bacteria using gel electrophoresis-nitrocellulose blotting

An optimized multilocus enzyme electrophoresis method, which involves polyacrylamide-agarose gel electrophoresis followed by electrophoretic transfers on nitrocellulose sheets, was developed for the analysis of enzyme polymorphism in several aerobic and anaerobic bacterial species including Staphylococcus aureus, Streptococcus pneumoniae, S. agalactiae, Klebsiella pneumoniae and K. oxytoca, Clostridium bifermentans and C. sordellii, and Prevotella bivia. Serial electrophoretic transfers (during 5-15 min each) from a single polyacrylamide gel could be achieved for most enzymes studied, and allowed an increased definition of enzyme bands on nitrocellulose as compared to migration gels. Four enzymes, which could not be blotted in such conditions, could still be stained in gels after blotting. Thus, the method allowed the combined analysis of several enzymes after a single gel electrophoresis separation. The analysis of enzyme polymorphism in the various species studied raised the interest of polymorphic loci such as esterase or glutamic-oxaloacetic transaminase for epidemiologic studies. The method characterized a genetic diversity of enzyme loci of S. pneumoniae higher than previously reported, and is thus convenient for the analysis of genetic relationships between related isolates. Since the present method reduces the tediousness of multilocus enzyme electrophoresis and requires experimental conditions that are not specific for the bacterial population studied, it may be proposed for rapid population genetics analysis of a wide variety of bacteria.     

Polyamine requirement for streptomycin action on protein synthesis in bacteria

Self-association between various heparan sulphate species and oligosaccharide fragments thereof have been studied by affinity chromatography. Polysaccharides or oligosaccharides were coupled to agarose and free chains were applied at low concentrations (less than or equal to 2 mg/ml) in 0.15 M NaCl to minimize self-association between free chains. The results show that the interaction may be specific. Heparan sulphate chains chiefly bind to gels substituted with cognate chains, i.e. the same kind or closely similar ones. Oligosaccharides of the general structure glucosamine-(iduronate/glucuronate-glucosamine)n--O--C(=CH2)--CHO were prepared by periodate oxidation/alkaline elimination and also coupled to agarose via the --CHO group. Cognate heparan sulphate chains were bound to this affinity matrix with the same affinity as in the case of heparan-sulphate--agarose. Free oligosaccharides were not bound to oligosaccharide-agarose, nor to the corresponding heparan-sulphate--agarose. Oligosaccharides of the same size and containing only iduronate were ineffective as affinity ligands. It is concluded that the segments comprising both iduronate and glucuronate may serve as contact zones in the heparan sulphate/heparan sulphate self-association and that the strength of binding is dependent on cooperative interactions between a number of such zones. The putative contact zones, as ligands on the matrix, showed an emerging lack of specificity as non-associating or unrelated and associating chains were bound to this gel. This is ascribed to a randomization of the contact zones which, in the polymeric chains, are placed in their proper register by the intervening (glucuronate-N-acetylglucosamine)n segments.     

Fluorescent staining of sialidases in polyacrylamide gel electrophoresis and ultrathin-layer isoelectric focusing

Polyacrylamide gels were stained with the sialidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid showing the activity of Vibrio cholerae and Clostridium sordellii sialidases in the gels after electrophoresis. With this fluorogenic method minimum sialidase activities of 5 microU could be determined. The sensitivity of this staining is about 10,000-fold higher compared to protein-staining with Coomassie brilliant blue. For the visualization of other proteins than sialidases the specific sialidase staining could be followed by a protein-staining method in the same gel.     

Characterization and expression of uterine and placental alkaline phosphatases in the mouse.

Alkaline phosphatase (ALP) is rapidly induced in the uterine subepithelial stroma after a natural or artificial decidual stimulus. During gestation ALP-specific activity peaked at Day 7 to 8 (Day 1 is day of detection of the copulation plug) followed by a rapid decline to control levels by Day 9. This elevation in enzyme activity was preceded by an 8-fold induction of a 2.6 kilobase (kb) mRNA. This mRNA was not preferentially localized to implantation sites. ALP activity was detected in the placenta at Day 9 and reached maximum specific activity at Day 19. The placental ALP was also encoded by a 2.6 kb mRNA. Uterine and placental ALPs were inhibited to the same extent by levamisole, L-tryptophan and homoarginine. The calculated Ki values for these inhibitors were not statistically different between the uterine and placental forms. Km values towards the substrate p-nitrophenylphosphate, however, were statistically different between the uterine and placental forms. Both uterine and placental ALPs were stimulated 3-4-fold by addition of 2 mM-Mg2+. Electrophoretic mobilities on SDS polyacrylamide gel, where the enzyme migrated as a single band, were the same. The uterine form, however, could be distinguished from the placental isoenzyme by separation on non-denaturing polyacrylamide gels; the uterine form had a single zone of activity which migrated with an intermediate mobility between the two zones of activity detected for the placental enzyme. These differences in mobility could be ascribed to the sialic acid content of the enzyme because treatment with neuraminidase resulted in the uterine and placental forms migrating with comparable but slower mobilities in non-denaturing gels.(ABSTRACT TRUNCATED AT 250 WORDS)     

A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis

Two-dimensional electrophoresis is a critical technique for proteome research, but currently available methods are not capable of resolving the >10,000 protein components in most eukaryotic proteomes. We have developed and demonstrated the utility of a novel solution isoelectrofocusing device and method that can reproducibly prefractionate cell extracts into well-defined pools prior to 2D PAGE on a scale directly compatible with the high sensitivity of proteome studies. A prototype device was used to separate metabolically radiolabeled Escherichia coli extracts in method optimization and proof-of-principle experiments. Samples were loaded into separation chambers divided by thin polyacrylamide gels containing immobilines at specific pH values and isoelectrically focused for several hours, which resulted in well-resolved fractions. Total recoveries in the fractionated samples were greater than 80% and most protein spots in the original sample were recovered after this prefractionation step. Nonideal behavior (precipitation/aggregation), typically encountered when unfractionated samples at high protein loads were applied directly to either narrow- or broad-range IPG gels, was dramatically reduced. Hence this approach allows increases in overall protein loads, resolution, and dynamic detection range compared with either alternative prefractionation methods or direct use of parallel narrow pH range gels without sample prefractionation. The pH ranges and number of fractions can be readily adapted to the requirements of specific types of samples and projects. This method should allow quantitative comparisons of at least 10,000 protein components on a series of narrow pH range gels, and protein detection limits are estimated to be 1000 molecules per cell when mammalian proteomes are fractionated into five or more pools.     

Assay of proteins by Lowry's method in samples containing 2-mercaptoethanol

A rapid method of screening chromatographic fractions has been developed for enzymes which metabolize fluorogenic substrates. Samples of the eluted fractions are applied to cellulose acetate gels and then incubated with the specific fluorogenic substrate. Fractions which possess enzymatic activity are visible as fluorescent spots when the gels are examined under long-wave ultraviolet light.