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PubMed2002

Characterization of polyacrylamide-stabilized Pfl phage liquid crystals for protein NMR spectroscopy.

A new polymer-stabilized nematic liquid crystal has been characterized for the measurement of biomolecular residual dipolar couplings. Filamentous Pf1 phage were embedded in a polyacrylamide matrix that fixes the orientation of the particles. The alignment was characterized by the quadrupolar splitting of the 2H NMR water signal and by the measurement of 1H-15N residual dipolar couplings (RDC) in the archeal translation elongation factor 1beta. Protein dissolved in the polymer-stabilized medium orients quantitatively as in media without polyacrylamide. We show that the quadrupolar splitting and RDCs are zero in media in which the Pf1 phage particles are aligned at the magic angle. This allows measurement of J and dipolar couplings in a single sample.

Acrylic Resins↗
PubMed1974

The preparation of crystalline human L-lactate--nicotinamide--adenine dinucleotide oxidoreductase isoenzyme 1 involvoing preparative polyacrylamide-gel electrophoresis.

A method is presented for the preparation of human heart lactate dehydrogenase (l-lactate-NAD(+) oxidoreductase; EC 1.1.1.27) isoenzyme 1; this involves the use of polyacrylamide-gel electrophoresis as a preparative step. The yield was about 10% with a final specific activity of 220 units/mg of protein, one unit being defined as the amount of enzyme catalysing the oxidation of 1mumol of NADH/min at 25 degrees C, in the presence of 0.33mm-pyruvate. The crystalline preparation contained less than 2% of the other isoenzymes, was homogeneous in the ultracentrifuge and showed only a trace of protein contamination on polyacrylamide-gel electrophoresis. Some properties of the crystalline isoenzyme are reported; E(1%) (1cm)=13.2 at 280nm, s(0) (20,w)=7.43S, pI=4.6, and the apparent K(m) for pyruvate=1.02x10(-4)m. The human isoenzyme and the isoenzyme from pig heart differ with respect to amino acid composition, electrophoretic mobility and solubility. It is possible that these differences do not involve the active site, or sites, but are due to changes in amino acid residues elsewhere in the molecule. The importance of purified human LDH-1 isoenzyme with regard to enzyme radioimmunoassay is emphasized.

Amino Acids↗
PubMed1979

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents.

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in specific ratios may be useful in the purification of adenylate cyclase and other intrinsic membrane proteins.

Adenylyl Cyclases↗
PubMed1981

Additional components of bovine heart cytochrome c oxidase demonstrated by high-resolution polyacrylamide-gel electrophoresis in the presence of chloral hydrate.

We have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) dissociates bovine heart cytochrome c oxidase. We have developed new procedures of polyacrylamide-gel electrophoresis in the presence of chloral hydrate that permit variation in the pH of the separation, and, by using these procedures, we have observed 15 components in preparations of the enzyme. This number contrasts with the eight bands that were seen on electrophoresis in the presence of SDS (sodium dodecyl sulphate) and urea. We have isolated material from these eight bands and have characterized each by electrophoresis in the presence of chloral hydrate. Twelve of the fifteen components that were seen by electrophoresis in chloral hydrate were identified as constituents of the eight bands seen by electrophoresis in the presence of SDS and urea. Two-dimensional electrophoretic separations confirmed these identifications ans showed that the other three components which were resolved as discrete bands by electrophoresis in the presence of chloral hydrate appeared to be diffusely present in the electrophoretic separations performed in the presence of SDS and urea, which suggested anomalous behaviour in that detergent. Trypsin treatment of cytochrome c oxidase caused total loss, as observed by electrophoretic separations in the presence of chloral hydrate, of a number of components. The trypsin-sensitive components included all of those that behaved anomalously in the presence of SDS and urea. Chloral hydrate is a potent non-ionic dissociating agent for cytochrome c oxidase and its use in polyacrylamide-gel electrophoresis, with variation in the pH of the gel, permits charge-dependent separations that should have general application in the analysis of membrane proteins.

Animals↗
PubMed1984

Separation of radiolabelled glycosaminoglycan oligosaccharides by polyacrylamide-gel electrophoresis.

Glycosaminoglycan oligosaccharides generated by treatment of biosynthetically radiolabelled dermatan sulphate and hyaluronic acid with chondroitin AC lyase or testicular hyaluronidase may be resolved into a series of discrete bands by polyacrylamide-gel electrophoresis. Bands were identified by fixation in glacial acetic acid containing 20% (w/v) 2,5-diphenyloxazole followed by fluorography. The bands represented glycans which differed in size by one disaccharide unit. For the larger oligosaccharides (decasaccharides and above) of similar charge: mass ratio, there was a linear relationship between electrophoretic mobility and log Mr. However, the smaller species showed anomalous migration patterns. Consideration of the structures of the fragments produced by the different enzyme treatments suggests that copolymeric and homopolymeric oligosaccharides may be separated by polyacrylamide-gel electrophoresis. There are many potential applications of this technique, foremost amongst them being studies on the molecular size heterogeneity and patterns of enzyme-mediated depolymerization of native glycosaminoglycan chains and investigations into rates of polymer chain elongation and post-polymerization modification reactions so essential to glycosaminoglycan function.

Chromatography, Gel↗
PubMed1988

Oligosaccharide mapping of heparan sulphate by polyacrylamide-gradient-gel electrophoresis and electrotransfer to nylon membrane.

A new method that we have called 'oligosaccharide mapping' is described for the analysis of radiolabelled heparan sulphate and other glycosaminoglycans. The method involves specific enzymic or chemical scission of polysaccharide chains followed by high-resolution separation of the degradation products by polyacrylamide-gradient-gel electrophoresis. The separated oligosaccharides are immobilized on charged nylon membranes by electrotransfer and detected by fluorography. A complex pattern of discrete bands is observed covering an oligosaccharide size range from degree of polymerization (d.p.) 2 (disaccharide) to approximately d.p. 40. Separation is due principally to differences in Mr, though the method also seems to detect variations in conformation of oligosaccharide isomers. Resolution of oligosaccharides is superior to that obtained with isocratic polyacrylamide-gel-electrophoresis systems or gel chromatography, and reveals structural details that are not accessible by other methods. For example, in this paper we demonstrate a distinctive repeating doublet pattern of iduronate-rich oligosaccharides in heparitinase digests of mouse fibroblast heparan sulphate. This pattern may be a general feature of mammalian heparan sulphates. Oligosaccharide mapping should be a valuable method for the analysis of fine structure and sequence of heparan sulphate and other complex polysaccharides, and for making rapid assessments of the molecular distinctions between heparan sulphates from different sources.

Chromatography, Gel↗
PubMed1988

Analysis of NADH dehydrogenases from plant [mung bean (Phaseolus aureus)] mitochondrial membranes on non-denaturing polyacrylamide gels and purification of complex I by band excision.

The present paper describes the analysis of plant mitochondrial NADH dehydrogenases using a recently developed non-dissociating gradient polyacrylamide-gel-electrophoresis technique [Kuonen, Roberts & Cottingham (1986) Anal. Biochem. 153, 221-226]. Solubilized mung-bean (Phaseolus aureus) submitochondrial particles were analysed on 3-22% (w/v) gradient polyacrylamide gels containing 0.1% Triton X-100 and stained for multiple NADH dehydrogenase activities. A rotenone-sensitive NADH dehydrogenase (Complex I) was identified on the basis of co-migration with the purified mammalian enzyme. The polypeptide composition of the plant enzyme was further analysed by band excision and SDS/polyacrylamide-gel electrophoresis.

Cytochrome Reductases↗
PubMed1983

Proteins of the kidney microvillar membrane; analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and crossed immunoelectrophoresis.

A microvillar fraction was prepared from human kidney cortex. This fraction was seven to 10 times enriched in aminopeptidases N and A, gamma-glutamyltransferase, dipeptidyl peptidase IV, neutral endopeptidase and alkaline phosphatase. Dipeptidyl peptidase IV activity of human renal microvilli could be inhibited by di-isopropylphosphorofluoridate and neutral endopeptidase activity by phosphoramidon. Nearly all the activity of aminopeptidases A and N could be removed from the membrane by treatment with papain, but only 19% and 33% of dipeptidyl peptidase IV and gamma-glutamyltransferase activities were released under the same conditions. Neutral endopeptidase and alkaline phosphatase were not solubilized by papain. Treatment with elastase gave results similar to papain, except that gamma-glutamyltransferase was not released. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions of microvilli revealed 36 polypeptide bands, 12 of which contained carbohydrate. A band of apparent Mr 130 000 was labelled with [3H]di-isopropylphosphorofluoridate and hence identified as dipeptidyl peptidase IV. Antibodies raised to human kidney microvilli produced 11 precipitates with detergent solubilized proteins and six with papain released proteins. Several of the precipitates were identified histochemically. Microvilli prepared from human kidney are very similar to microvilli from pig and rabbit kidney with respect to enzymology, response to papain treatment, sodium dodecyl sulphate-polyacrylamide gel patterns and immunochemistry.

Adolescent↗
PubMed1999

Importance of sodium dodecyl sulfate pore-graduated polyacrylamide gel electrophoresis in the differential diagnostic of Balkan nephropathy.

Balkan nephropathy (BN) is an endemic disease, which leads to end-stage renal failure and artificial renal replacement therapy. Pathologically it is characterized by progressive interstitial nephritis in a large population of villages situated in the proximity of a bend of the Danube up to a distance of 100 km from the river in several parts of Bulgaria, Romania, and the former Yugoslavia. The urinary proteins of 19 patients with BN from the region of Vratza, Bulgaria were examined using ultrathin layer sodium dodecyl sulfate (SDS) pore-graduated polyacrylamide gel electrophoresis (PAGE) and silver staining. The documentation of urinary proteins pattern was performed using laser densitometry and consecutive electronic processing for the purpose of characterizing and quantifying protein excretion. Our results show that the proteinuria of BN is predominantly tubular, consisting of low molecular weight species (10-65 kilodaltons). The amount of tubular protein changes with the progression of the disease. SDS-polyacrylamide gel electrophoresis (PAGE) is a diagnostic method for early diagnosis of tubular failure in BN. Using our method of SDS-PAGE, tubular failure can be detected even at a total protein concentration below 0.1 g/L and when the serum creatinine concentration is normal. Additionally, our method of SDS-PAGE supports the differentiation of BN from glomerular disease.

Aged↗
PubMed2006

Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis.

Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn2+ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3beta, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC50 value of 1.6 microM in the presence of 0.10 mM ATP.

Cell Line, Tumor↗
PubMed1976

Serum protein fractions from children of differing nutritional status analysed by polyacrylamide gel electrophoresis and electroimmunoassay.

1. The nutritional status of children showing no clinical signs of malnutrition, from the University School of Khon Kaen, Khon Kaen province, north-east Thailand and from two villages nearby, was tested. The children were grouped according to their body-weight expressed as a percentage of expected weight-for-height (Harvard standards (Stuart & Stevenson, 1959), as given by Jelliffe (1966)). 2. The differing prealbumin concentrations indicated that nutritional status differed between the groups. 3. The urinary urea: creatinine ratio was significantly lower in the village children compared with the children from Khon Kaen, indicative of the higher dietary protein intake of the latter. 4. alpha1-Acid glycoprotein and the first 'post-albumin peak' (obtained by polyacrylamide gel electrophoresis of serum and containing mainly mainly Gc-globulin, alpha1-antichymotrypsin and alpha1-B-glycoprotein) were found to be significantly higher in the village children compared with children from Khon Kaen. 5. The three main proteins of the first 'post-albumin peak' from polyacrylamide gel electrophoresis of serum were tested separately using the electroimmunoassay method. There was no significant difference in Gc-globulin between the children from Khon Kaen and the village children. The concentration of alpha1-B-glycoprotein from those Khon Kaen children whose body-weight was more than 95% expected weight-for-height was significantly lower compared with that of village children, alpha1-Antichymotrypsin concentration was significantly higher in serum from Khon Kaen children than in serum from village children.

Blood Protein Electrophoresis↗
PubMed2004

Comparative analysis of TCR-gamma gene rearrangements by Genescan and polyacrylamide gel-electrophoresis in cutaneous T-cell lymphoma.

Demonstrating T-cell clonality has become an important approach supporting a diagnosis of malignant T-cell neoplasms. A comparative study between Genescan analysis, polyacrylamide gel and agarose gel electrophoresis in visualizing T-cell receptor gamma gene rearrangement was performed on 25 biopsy specimens from 18 patients with different forms of cutaneous T-cell lymphomas. Clonality was detected in 17 biopsy specimens when PCR products were evaluated by Genescan analysis. Seventeen showed discrete bands when visualized in polyacrylamide gel and 14 cases were clonal when visualized with agarose gel. In five cases, a clonal population was seen in the gels, but not with Genescan. On sequencing the PCR products we demonstrated non-clonality of these five samples. Our results confirm that PCR-Genescan is a useful, reliable and specific screening method for detecting dominant clones in patients with T-cell lymphoma.

Electrophoresis, Polyacrylamide Gel↗
PubMed1977

A rapid and effecient method for the isolation of proteins from polyacrylamide cells.

A procedure is described for the rapid and efficient electrophoretic elution of protein from polyacrylamide gels which is then collected in a dialysis bag tied to the end of a tube containing the gel slices. To illustrate the method a heterogeneous preparation of alkaline phosphatase was used from which a single homogeneous component was isolated in six hours with a recovery of 86%. The eluted protein is collected in a volume which can easily be kept below 1.5 ml, thus eliminating the need for subsequent concentration. The method has also been used successfully in two other systems in which a human lung tumor-associated antigen and glycogen synthetase from yeast were isolated. Since the method utilizes a standard analytical gel electrophoresis apparatus with no modifications or accessories, it should be immediately applicable for the isolation of many different proteins from polyacrylamide gels.

Alkaline Phosphatase↗
PubMed1979

Preparation polyacrylamide-agarose gel electrophoresis of proteins and RNAs by the use of new device.

Quantitatively reproducible results were obtained by using a new device for preparation gel electrophoresis combined with polyacrylamide-agarose composite gel. When an adequate gel-buffer system was selected according to the procedure described in this paper, proteins and RNA's were well separated and recovered. The new device for preparative gel electrophoresis and the method for preparation of polyacrylamide-agarose composite gel are presented together with the elution profiles of the recovered substances.

Blood Proteins↗
PubMed1987

A sensitive urea-silver stain method for detecting trace quantities of separated proteins in polyacrylamide gels.

An ultrasensitive method using a urea-silver staining procedure to detect trace quantities of proteins in polyacrylamide gels (PAGE) is described. This technique is sensitive enough to detect picogram quantities of proteins resolved on sodium dodecyl sulfate-polyacrylamide gels. The major advantages of our method are that it provides a clear background and it is more sensitive than other techniques allowing it to substitute for radioisotopic techniques in some cases.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Polyacrylamide-chitosan hydrogels: in vitro biocompatibility and sustained antibiotic release studies.

Controlled drug delivery is gaining importance over the conventional methods of drug administration because of its inherent benefits. Self-regulated release from the delivery vehicle may enhance drug potency with a sustained action. The present study describes a novel hydrogel blend of polyacrylamide with chitosan for controlled delivery of antibiotics. Hydrogel was synthesized by cross-linking acrylamide-chitosan mixture (8:2 v/v) with N,N' methylene bisacrylamide. Hydrogel was characterized for surface morphology, hydrophilicity, pH-dependent swelling properties, cytotoxicity, and control release properties. Scanning electron microscopy (SEM) revealed the macroporous surface morphology of the matrix with average pore size at 104 +/- 7.61 mu. Hydrogel was found to be highly hydrophilic as assessed by octane contact angle (154.5 + 0.572) measurement. Hydrogel showed no cytotoxic effects on NIH3T3 and HeLa cells up to 40% of extract concentrations as determined by MTT and neutral red assay. This showed hydrogel biocompatibility and thus absence of deleterious effects of the hydrogel on cell viability and functionality. Hydrogels did not show any pH-dependent swelling profile, and they swelled considerably to achieve a swelling ratio of approximately 16.0 at the end of 24 hr. Amoxicillin was incorporated in the hydrogel matrix as a candidate antibiotic for release studies. In vitro release studies of amoxicillin revealed the sustained nature of delivery and matrix released 56.47 + 1.12% and 77.096 + 1.72% of amoxicillin at the end of 24 and 75 hr, respectively. Although in vivo studies are awaited, the present study provides enough documentation to consider polyacrylamide-chiotsan hydrogel as a possible candidate for controlled delivery of antibiotics.

3T3 Cells↗
PubMed1991

Electrophoretic elution of macromolecules from polyacrylamide or agarose gels.

A method for elution of micrograms of macromolecules from polyacrylamide and agarose gels is described. The recoveries were greater than 90% with three different macromolecules tested (28 to 360 kDa). An amount as small as 1 microgram of human serum albumin was eluted from polyacrylamide gel with 90% recovery. The eluted material is collected into a small chamber the size of which can be changed as required. Elution and concentration are achieved simultaneously and in one step under mild conditions. Sterile eluates can be obtained, if the apparatus is constructed under sterile conditions.

Apolipoproteins A↗
PubMed1999

Experimental 3D dosimetry around a high-dose-rate clinical 192Ir source using a polyacrylamide gel (PAG) dosimeter.

It is well known that the experimental dosimetry of brachytherapy sources presents a challenge. Depending on the particular-dosimeter used, measurements can suffer from poor spatial resolution (ion chambers), lack of 3D information (film) or errors due to the presence of the dosimeter itself distorting the radiation flux. To avoid these problems, we have investigated the dosimetry of a clinical 192Ir source using a polyacrylamide gel (PAG) dosimeter. Experimental measurements of dose versus radial distance from the centre of the source (cross-line plots) were compared with calculations produced with a Nucletron NPS planning system. Good agreement was found between the planning system and gel measurements in planes selected for analysis. Gel dosimeter measurements in a coronal plane through the phantom showed a mean difference between measured absorbed dose and calculated dose of 0.17 Gy with SD = 0.13 Gy. Spatially, the errors at the reference point remain within one image pixel (1.0 mm). The use of polymer gel dosimetry shows promise for brachytherapy applications, offering complete, three-dimensional dose information, good spatial resolution and small measurement errors. Measurements close to the source, however, are difficult, due to some of the limiting properties of the polyacrylamide gel.

Acrylic Resins↗