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PubMed2004

Chemiluminescent image detection of haptoglobin phenotyping after polyacrylamide gel electrophoresis.

The development of an enhanced chemiluminescence detection method for the rapid detection of haptoglobin phenotyping after polyacrylamide gel electrophoresis is described in this paper. The enhanced chemiluminescence detection is based upon chemiluminescent reaction between luminol and hydrogen peroxide. Increased sensitivity and dynamic range are achieved by employing ammonium persulfate to enhance the chemiluminescence signal. Detection of haptoglobin phenotypes in human blood serum was easily achieved even without the addition of hemoglobin. Different polyacrylamide gel electrophoresis results were found between pure serum and hemoglobin-supplemented serum. Applying the suggested enhanced chemiluminescence detection, the original combining forms of haptoglobin and hemoglobin can be detected. The linear range of haptoglobin is 0.1-13.3 microg/mL, with a detection limit of 1.21 ng (sample loading volume 15 microL). Other proteins, such as catalase and ferritin, can also be detected using enhanced chemiluminescence detection. All detections after polyacrylamide gel electrophoresis were completed within 15 min. The proposed detection is very fast, compared to traditional methods using staining detection (minutes versus hours).

Ammonium Sulfate↗
PubMed2004

Surface modification of the channels of poly(dimethylsiloxane) microfluidic chips with polyacrylamide for fast electrophoretic separations of proteins.

The electrophoresis of proteins was investigated using poly(dimethylsiloxane) (PDMS) microfluidic chips whose surfaces were modified with polyacrylamide through atom-transfer radical polymerization. PDMS microchips were made using a glass replica to mold channels 10 microm high and 30 microm wide, with a T-intersection. The surface modification of the channels involved surface oxidation, followed by the formation of a self-assembled monolayer of benzyl chloride initiators, and then atom-transfer radical polymerization to grow a thin layer of covalently bonded polyacrylamide. The channels filled spontaneously with aqueous buffer due to the hydrophilicity of the coating. The resistance to protein adsorption was studied by open-channel electrophoresis for bovine serum albumin labeled with fluorophor. A plate height of 30 microm, corresponding to an efficiency of 33 000 plates/m, was obtained for field strengths from 18 to 889 V/cm. The lack of dependence of plate height on field strength indicates that there is no detectable contribution to broadening from adsorption. A 2- to 3-fold larger plate height was obtained for electrophoresis in a 50-cm polyacrylamide-coated silica capillary, and the shape of the electropherogram indicated the efficiency is limited by a distribution of species. The commercial capillary exhibited both reversible and irreversible adsorption of protein, whereas the PDMS microchip exhibited neither. A separation of lysozyme and cytochrome c in 35 s was demonstrated for the PDMS microchip.

Acrylic Resins↗
PubMed1991

Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis.

We have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their [32P]DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase beta were found to be similar under both in vitro and in situ conditions. With 3'-terminally matched and mismatched [32P]DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. In addition, use of matched and mismatched DNA primers permitted the uncoupling of mismatch excision and chain extension steps. Activities first detected in nondenaturing activity gels as either multifunctional or multimeric enzymes were also identified in denaturing activity gels, and assignment of activities to specific polypeptides suggested subunit composition. Furthermore, DNA substrates cast within polyacrylamide gels were successfully modified by the exogenous enzymes polynucleotide kinase and alkaline phosphatase before and after in situ detection of E. coli DNA ligase activity, respectively. Several restriction endonucleases and the tripeptide (Lys-Trp-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.

Base Sequence↗
PubMed2002

Molecular alignment of denatured states of staphylococcal nuclease with strained polyacrylamide gels and surfactant liquid crystalline phases.

Residual dipolar couplings reflect the orientation of vectors between pairs of magnetic nuclei relative to a unique set of molecular axes. Thus, unlike NOEs and scalar couplings, dipolar couplings provide access to long-range structural information. A prerequisite for measurement of these NMR parameters is imposition of a weak net alignment, most simply by forcing the macromolecules to tumble in an asymmetric environment that restricts some orientations more than others. In this report, several denatured forms of staphylococcal nuclease are aligned by using compressed and stretched polyacrylamide gels, a nonionic type of lipid bilayer disk or bicelle, and a liquid crystalline phase formed by a cationic lipid. All three types of media can be used at high urea concentrations. While polyacrylamide gels and bicelles produce similar alignment tensors through steric interactions, a liquid crystalline phase of cetylpyridinium bromide aligns denatured nuclease along a different set of axes, presumably through electrostatic effects. The analysis of residual dipolar couplings collected with two different alignment tensors may permit the calculation of ensembles of conformations. The dipolar couplings observed for staphylococcal nuclease denatured with urea, by low pH or by deletion of residues from both termini, suggest that all denatured forms share a common "topology", one which has been shown previously to be native-like. Although SDS/nuclease complexes give sharp and disperse (1)H-(15)N correlation spectra, only small couplings are observed in strained polyacrylamide gels.

Acids↗
PubMed2005

Lipid bilayers on polyacrylamide brushes for inclusion of membrane proteins.

The ability of neutral polymer cushions to support neutral lipid bilayers for the incorporation of mobile transmembrane proteins was investigated. Polyacrylamide brush layers were grown on fused silica using atom-transfer radical polymerization to provide polymer layers of 2.5-, 5- and 10-nm thickness. Lipid bilayers composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) were formed by vesicle fusion onto bare fused silica and onto each of the polyacrylamide layers. Bilayer fluidity was assessed by the diffusion of a probe, NBD-labeled phosphatidylcholine, using fluorescence recovery after photobleaching. A transmembrane protein, the human delta-opioid receptor, was inserted into each lipid bilayer, and its ability to bind a synthetic ligand, DPDPE, cyclic[2-d-penicillamine, 5-d-penicillamine]enkephalin, was detected using single-molecule fluorescence spectroscopy by labeling this ligand with a rhodamine dye. The transmembrane protein was observed to bind the ligand for all bilayers tested. The protein's electrophoretic mobility was probed by monitoring the fluorescence from the bound ligand. The 5-nm polyacrylamide thickness gave the fastest diffusion for the fluorescent lipid probe (D(1) = 2.0(+/-1.2) x 10(-7) and D(2) = 1.2(+/-0.5) x 10(-6) cm(2)/s) and also the largest electrophoretic mobility for the transmembrane protein (3 x 10(-8) cm(2)/V.s). The optimum in polymer thickness is suggested to be a tradeoff between decoupling from the substrate and increasing roughness of the polymer surface.

Acrylic Resins↗
PubMed1976

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.

Electrophoresis, Polyacrylamide Gel↗
PubMed1999

Improved method for non-denaturing polyacrylamide gradient gel electrophoresis for detection of small-sized LDL produced during postprandial hypertriglyceridaemia.

A reliable method for detection of small-sized low-density lipoprotein (LDL) produced during transient hypertriglyceridaemia induced by ingestion of fat is described. Electrophoresis using a commercial non-denaturing 2.0-16% polyacrylamide gradient gel is commonly utilized to determine the size of LDL particles, but it failed to detect a minor change in LDL size induced during postprandial hypertriglyceridaemia. Detection of small-sized LDL was achieved by adjusting the polyacrylamide concentration. Electrophoresis using a non-denaturing 1.5-10% polyacrylamide gradient gel gave the best resolution for detecting small-sized LDL induced during postprandial hypertriglyceridaemia. This improved method may be useful for elucidating the underlying mechanism of production of small-sized LDL (small dense LDL) in subjects with chronic hypertriglyceridaemia.

Dietary Fats↗
PubMed1994

Molecular sequestration stabilizes CAP-DNA complexes during polyacrylamide gel electrophoresis.

The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids. Often it is found that complexes that are short-lived in free solution (t1/2 of the order of minutes) persist for hours under the conditions of gel electrophoresis. We have investigated the influence of polyacrylamide gels on the pseudo first-order dissociation kinetics of complexes containing the E.coli cyclic AMP receptor protein (CAP) and lactose promoter DNA. Within the gel matrix, kdiss decreased with increasing [polyacrylamide] and the order of the reaction was changed. In free solution, kdiss was proportional to [DNA]2, while in 5% gels, kdiss was proportional to [DNA]0.3. In gels of [polyacrylamide] > or = 10%, kdiss was nearly independent of [DNA] until fragment concentrations exceeded 0.1 microM. Even in the absence of competing DNA, kdiss(gel) < kdiss(solution). These results suggest that the lifetime of CAP-DNA complexes in free solution is limited by their encounter frequency with molecules of DNA or with protein-DNA complexes; some or all of the stabilization observed in gels may be due to a reduction in this frequency.

Cyclic AMP Receptor Protein↗
PubMed1985

Antigenic alteration of contaminating lipopolysaccharide during extraction of Escherichia coli outer-membrane proteins from polyacrylamide gels.

An antiserum raised to the ferric enterobactin receptor protein of Escherichia coli, isolated from SDS-polyacrylamide gels, contained high-titre antibodies to the lipopolysaccharide (LPS) of E. coli O111. This antiserum was used to show that proteins dissected from polyacrylamide gels can be contaminated with comigrating LPS at levels below those detectable by very sensitive silver staining methods. Using this antiserum it was also shown that the procedures used to extract proteins from polyacrylamide gels can alter the molecular structure and, consequently, the antigenic properties of the contaminating LPS.

Antigens, Bacterial↗
PubMed1991

The resolution of bacteroides lipopolysaccharides by polyacrylamide gel electrophoresis.

The lipopolysaccharides (LPS) of the 10 species of the genus Bacteroides (sensu stricto) were extracted by the proteinase K method and their resolution compared by several methods of polyacrylamide gel electrophoresis (PAGE). These included sodium dodecyl sulphate (SDS)-PAGE with and without urea, polyacrylamide gradient gels and Tricine [N-Tris (hydroxymethyl) methyl glycine]-SDS-PAGE. The original discontinuous system showed good resolution of LPS from B. thetaiotaomicron, B. caccae and B. ovatus and this was enhanced by urea; B. vulgatus showed a typical ladder pattern associated with repeating polysaccharide units of the O side chains. The LPS profiles of the other species, including B. fragilis, were poorly resolved; the majority of components migrated with the leading edge of the wave front. The resolution of the LPS of these species was marginally improved with gradient gels but the majority of components were separated only within the regions of high polyacrylamide concentration. The Tricine-SDS system was consistently superior to the other methods, with excellent resolution of the LPS profiles of all Bacteroides species.

Bacteroides↗
PubMed2004

Estimates of lifetime-absorbed daily doses from the use of personal-care products containing polyacrylamide: a Monte Carlo analysis.

Estimates of the lifetime-absorbed daily dose (LADD) of acrylamide resulting from use of representative personal-care products containing polyacrylamides have been developed. All of the parameters that determine the amount of acrylamide absorbed by an individual vary from one individual to another. Moreover, for some parameters there is uncertainty as to which is the correct or representative value from a range of values. Consequently, the parameters used in the estimation of the LADD of acrylamide from usage of a particular product type (e.g., deodorant, makeup, etc.) were represented by distributions evaluated using Monte Carlo analyses.((1-4)) From these data, distributions of values for key parameters, such as the amount of acrylamide in polyacrylamide, absorption fraction, etc., were defined and used to provide a distribution of LADDs for each personal-care product. The estimated total acrylamide LADD (across all products) for males and females at the median, mean, and 95th percentile of the distribution of individual LADD values were 4.7 x 10(-8), 2.3 x 10(-7), and 7.3 x 10(-7) mg/kg/day for females and 3.6 x 10(-8), 1.7 x 10(-7), and 5.4 x 10(-7) mg/kg/day for males. The ratio of the LADDs to risk-specific dose corresponding to a target risk level of 1 x 10(-5), the acceptable risk level for this investigation, derived using approaches typically used by the FDA, the USEPA, and proposed for use by the European Union (EU) were also calculated. All ratios were well below 1, indicating that all the extra lifetime cancer risk from the use of polyacrylamide-containing personal-care products, in the manner assumed in this assessment, are well below acceptable levels. Even if it were assumed that an individual used all of the products together, the estimated LADD would still provide a dose that was well below the acceptable risk levels.

Acrylamide↗
PubMed1975

Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of the human erythrocyte membrane.

A two-dimensional polyacrylamide gel electrophoresis technique has been developed, improving the analytical separation of some proteins and glycoproteins of the human erythrocyte membrane. Freshly prepared membranes are totally solubilized, subjected to dodecylsulfate--polyacrylamide gel electrophoresis in the first dimension, followed by electrophoresis in the second dimension, using a detergent-free polyacrylamide gradient gel. By this method the proteins of the human erythrocyte membrane could be resolved into a two-dimensional pattern, which has been shown to be highly reproducible with respect to various blood-groups and within one blood-group from specimen to specimen. The method enables especially the investigation of the hydrophobic and very likely integrated membrane proteins and glycoproteins. Thus, band III[Fairbanks, G., Steck, Th. & Wallach, D. F. H., Biochemistry, 10, 2606--2617 (1971)] could be shown to consist of five proteins, one of them being the major glycoprotein of the human erythrocyte membrand. The two spectrin bands differed considerably in their two-dimensional patterns. The value of the given method for the investigation of membrane defects, which may be linked with various diseases of human erythrocytes, could be demonstrated in the case of two patients suffering from congenital dyserythropoetic anaemia.

Blood Proteins↗
PubMed1977

Calf-ovary protein kinases dependent on adenosine 3':5' -monophosphate. Analysis by electrophoresis and electro-focusing on polyacrylamide get.

High resolving power and quantitative application polyacrylamide-gel electrophopresis at various pore sizes and electrofocusing provide resolution of a calf-ovarian protein-kinase system at an increased level of magnification, as well as optimal preparative routes. Three protein kinases dependent on adenosine 3':5' -monophosphate are distinguished by polyacrylamide gel electrophoresis in calf ovarian cytosol. These enzymes which are observed in the pH range 7.5--10.2, appear to be aggregates of a commonsubmit or monomer. The three kinases are, by the criteria of polyacylamide gel electrophoresis, distinct from three adenosine-3':5' -monophosphate-binding proteins found in the calf ovarian system. Analysis by electrofocusing on polyacrylamide gel shows that conventionally purified preparations of the major kinase of cytosol contain an overwhelming majority of contaminant proteins.

Animals↗
PubMed1996

A comparison between starch and polyacrylamide gels for the analysis of Listeria monocytogenes using multilocus enzyme electrophoresis.

Forty-seven Listeria monocytogenes isolates were analysed using multilocus enzyme electrophoresis in two laboratories. Both assayed for the same six enzymes, but one used a starch gel method and the other polyacrylamide gels. The starch gel method distinguished six electrophoretic types whereas the polyacrylamide gel method produced 17 different electrophoretic types. The polyacrylamide gel method was more discriminatory than the starch gel method.

Adenylate Kinase↗
PubMed1990

Comparisons of Pasteurella multocida lipopolysaccharides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine relationship between group B and E hemorrhagic septicemia strains and serologically related group A strains.

Lipopolysaccharides (LPSs) purified from 16 reference somatic serotypes of Pasteurella multocida were examined and compared by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of LPS patterns in a gel was optimum when sample wells were cast separately from the stacking gel and the running gel consisted of 15% T (total monomer) polyacrylamide and 4 M deionized urea. Band patterns of P. multocida LPSs in a gel differed from control Salmonella minnesota wild-type and core mutant LPSs. Although the band patterns and mobilities of LPSs from some P. multocida reference serotypes were similar, none were identical. Evidence for O antigens similar to those produced by enterobacteria was not observed. Proteinase K digestion of whole P. multocida cells resulted in LPS band patterns similar to those of purified LPS. The presence or absence of a capsule on a strain had no major influence on band patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparisons of LPS patterns of group B and E hemorrhagic septicemia strains with those of serologically related group A strains of P. multocida indicated that they were similar. Typing antisera made with purified serotype 2 or 5 LPS reacted with electroblots of all these strains. However, the reactions did not distinguish strains as being serotype 2 or 5.

Animals↗
PubMed1974

Newcastle disease virus-specific RNA: polyacrylamide gel analysis of single-stranded RNA and hybrid duplexes.

Newcastle disease virus-specific [(3)H]uridine-labeled 18S RNA was resolved by polyacrylamide gel electrophoresis into several components with molecular weights from 450,000 to 840,000. The analysis of 35 and 24S virus-specific RNA also revealed several components in each sedimentational class. The conversion of 18S RNA into double-stranded form by hybridization with an excess of unlabeled virion RNA improved the resolution in polyacrylamide gels and revealed at least six distinct components. The same six classes of hybrid duplexes were revealed when (32)P-labeled 50S virion RNA was hybridized with an excess of 18S RNA. The applicability of polyacrylamide gel electrophoresis of hybrid duplexes to the analysis of viral genome structure is discussed.

Animals↗
PubMed1975

Polyacrylamide gel electrophoresis of intact bacteriophage T4D particles.

A method for the electrophoresis of intact bacteriophage T4D particles through polyacrylamide gels has been developed. It was found that phage particles will migrate through dilute polyacrylamide gels (less than 2.1%) in the presence of a low concentration of MgCl2. As few as 5 x 10(9) phage particles can be seen directly as a light-scattering band during the course of electrophoresis. The band can also be detected by scanning gels at 260 to 265 nm or by eluting viable phage particles from gel slices. A new mutant (eph1) has been identified on the basis of its decreased electrophoretic mobility compared with that of the wild type; mutant particles migrated 14% slower than the wild type particles at pH 8.3 and 35% slower at pH 5.0. The isoelectric points of both the wild type and eph1 mutant were found to be between pH 4.0 and 5.0. Particles of T4 with different head lengths were also studied. Petite particles (heads 20% shorter than normal) migrated at the same rate as normal-size particles. Giant particles, heterogenous with respect to head length (two to nine times normal), migrated faster than normal-size particles as a diffuse band. This diffuseness was due to separation within the band of particles having mobilities ranging from 8 to 35% faster than those of normal-size particles. These observations extend the useful range of polyacrylamide gel electrophoresis to include much larger particles than have previously been studied, including most viruses.

Coliphages↗
PubMed1982

Core particles of hepatitis B virus and ground squirrel hepatitis virus. I. Relationship between hepatitis B core antigen- and ground squirrel hepatitis core antigen-associated polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping.

The relationships among the core antigen polypeptides of hepatitis B virus (HBV) and ground squirrel hepatitis virus (GSHV) were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping. The major core antigen polypeptides of liver-derived HBV (p22) and GSHV (p20.5) shared 56% of the spots in their peptide maps. Comparison of hepatitis B core antigen (HBcAg) p19 or ground squirrel hepatitis core antigen (GSHcAg) p16.5 with their respective major polypeptides indicated that these components probably resulted from cleavage of the major polypeptide of each virus. Other polypeptides smaller than the major component of each virus were often faint on polyacrylamide gels and probably resulted from the cleavage or degradation of components larger than p22 of HBcAg or p20.5 of GSHcAg, since their peptide maps contained spots unique to these high-molecular-weight components. p26 of GSHcAg and p27.5 of HBcAg shared approximately two-thirds of the spots on their peptide maps with those of their respective major core polypeptides. Furthermore, p37.5 of GSHcAg and p40 of HBcAg shared about 60% homology with their respective major polypeptides, and also shared many of the spots that were unique to p26 of GSHcAg or p27.5 of HBcAg but were not found in the peptide map of their respective core antigen polypeptides. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis bands larger than 40,000 daltons were variably present, and peptide mapping indicated that these were aggregates of various smaller core antigen-associated polypeptides. The results suggest that p40 of HBcAg and p37.5 of GSHcAg are the largest unique polypeptides in these core particles, and that they are encoded for by the genome of each virus. That a subset of the spots unique to p40 or p37.5 was also found in p27.5 of HBcAg or p26 of GSHcAg, respectively, as compared to the major core polypeptides, also suggests that p27.5 and p26 are unique proteins encoded by the genome of each virus. It is proposed that the core antigen gene of each virus is larger than that which would encode the major polypeptide of each virus, and that the genetic organizations of the core genes of HBV and GSHV are very similar.

Amino Acid Sequence↗