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PubMed1991

Heterogeneity of HDL in crossed immunoelectrophoresis with continuous gradient polyacrylamide gel in the first dimension and one or two antibody layers in the second dimension.

We describe a new method of immunoelectrophoresis with a continuous gradient polyacrylamide gel in the first dimension and an agarose-dextran gel in the second dimension with one or two layers of antibody. The use of a polyacrylamide gel in the first dimension allows better resolution of lipoproteins than with crossed immunoelectrophoresis using agarose gel in both dimensions. The use of two layers of antibody in the second dimension also enhances the specificity of characterization and the resolution of the separation. Thus, using a layer of anti-apo A-I combined with a layer of anti-apo A-II, three particles containing only apo A-I and three containing both apo A-I and A-II could be separated.

Adult↗
PubMed2000

Enantiomeric separations of cationic and neutral compounds by capillary electrochromatography with monolithic chiral stationary phases of beta-cyclodextrin-bonded negatively charged polyacrylamide gels.

Enantiomeric separation by capillary electrochromatography with beta-cyclodextrin-bonded negatively charged polyacrylamide gels was examined. The columns used are capillaries filled with a negatively charged polyacrylamide gel, a so-called monolithic stationary phase, to which allyl carbamoylated beta-CD (AC-beta-CD) derivatives covalently bind. The capillary wall is activated first with a bifunctional reagent to make the resulting gel bind covalently to the inner surface of the fused-silica tubing. Enantiomeric separations of 15 cationic compounds were achieved using the above-mentioned columns and mobile phases of 200 mmol l(-1) Tris-300 mmol I(-1) boric acid buffer (pH 7.0 or 9.0) or 200 mmol l(-1) Tris-300 mmol l(-1) boric acid buffer (pH 7.0) containing an achiral crown ether (18-crown-6). Enantiomeric separations of two neutral compounds were also achieved using 200 mmol l(-1) Tris-300 mmol l(-1) boric acid buffer (pH 9.0) as a mobile phase. High efficiencies of up to 150,000 plates m(-1) were obtained. Both the within- and between-run reproducibilities of retention time and separation factor were good. The reproducibilities of retention time and separation factor for three different columns prepared from a different batch of monomers were acceptable. The gel-filled capillaries were stable for at least 3 months with intermittent use, utilizing the mobile phase of 200 mmol I(-1) Tris-300 mmol I(-1) boric acid buffer (pH 9.0).

Acrylic Resins↗
PubMed2000

Mobility and activation energy of single-stranded DNA in denaturing cross-linked polyacrylamide slab gels.

An original apparatus based on laser-induced fluorescence detection is presented. One lane migration combined to four equidistant detection points allows the study of the dynamics of DNA bands during electrophoresis. We focus this article on the study of the mobility of DNA sequencing fragments as a function of temperature; mobility is determined in 4% T, 5% C and 4.3% T, 5% C cross-linked polyacrylamide gels at an electric field of 45 V/cm [T=(g acrylamide+g N,N'-methylenebisacrylamide)/100 ml solution; C=g N,N'-methylenebisacrylamide/% T]. Activation energy has been investigated under these experimental conditions with a temperature varying from 25 to 50 degrees C. The activation energy for migration through the cross-linked polyacrylamide gel decreases with fragment length under our experimental conditions and it varies along the migration.

DNA, Single-Stranded↗
PubMed2001

Enantiomeric separations of primary amino compounds by capillary electrochromatography with monolithic chiral stationary phases of chiral crown ether-bonded negatively charged polyacrylamide gels.

A novel enantiomeric separation method by capillary electrochromatography with chiral crown ether-bonded negatively charged polyacrylamide gels is presented. Two kinds of chiral crown ether derivatives, (+)-tetraallyl 18-crown-6 carboxylate and (+)-18-crown-6 tetracarboxylic acid 2-allyl ester were synthesized and allowed to covalently bind to a negatively charged polyacrylamide gel, a so-called monolithic stationary phase, respectively. The gel was placed in fused-silica tubing, the walls of which had been activated with a bifunctional reagent to make the resulting gel bind covalently to the inner surface. Enantiomeric separations of 12 primary amino compounds were achieved using these columns and mobile phases of 200 mM triethanolamine-300 mM boric acid buffers with high efficiencies of up to 135000 plates m(-1). Both the within- and between-run reproducibilities of retention time and separation factor were good. The reproducibilities of retention time and separation factor for three different columns prepared from a different batch of monomers were acceptable. The gel-filled capillaries were stable for at least 13 months with intermittent use for 3 months followed by storage at room temperature for 10 months. The result of the optical purity test of alanine-2-naphthylamide is also described.

Amines↗
PubMed1987

High-performance sodium dodecyl sulfate polyacrylamide gel capillary electrophoresis of peptides and proteins.

High-performance capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been developed for the separation and molecular weight (MW) determination of peptides and proteins. In this work, acrylamide was polymerized in fused-silica capillaries of 75 microns I.D. and 10 or 20 cm length. On-line UV detection was employed by placing the capillary in the optical path of the detector. Rapid separations of myoglobin fragments and of a mixture of standard proteins were demonstrated with efficiencies of the order of 40,000 plates for a column, 20 cm in length. Linear plots of log MW vs. mobility for the SDS protein complexes were observed. Variation in the percentage monomer composition (% T) of the polyacrylamide yielded the expected common intercept of log mobility vs. % T, confirming the size separation mechanism in the gel capillaries. The separation of the A and B chains of insulin with a 10-cm column in less than 10 min was also demonstrated.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Enzyme cytochemical staining of individual cells with the use of a polyacrylamide carrier. Studies on the synthetizing reaction technique, the indigogenic method, the metal salt method, the post-azo-coupling technique, and the tetrazolium salt technique.

A recently developed method for the (quantitative) demonstration of glucose-6-phosphate dehydrogenase activity in individual cells with the use of a polyacrylamide carrier has been extended for other enzyme cytochemical techniques. Isolated hepatocytes have been incorporated in the matrix of a thin transparent polyacrylamide gel prior to incubation in a cytochemical medium. The techniques which have been applied are the synthetizing reaction technique for glycogen phosphorylase, the indigogenic method for nonspecific esterase, the metal salt method for glucose-6-phosphatase, the post-azo-coupling technique for acid phosphatase, and the tetrazolium salt technique for succinate and lactate dehydrogenase activities. In all cases a few major problems which occur in the cytochemistry on single cells seem to be solved. The morphology is very well preserved, the final reaction product seems to be precipitated at the expected site of enzyme activity and the coloured end-product is highly specific for the enzyme activity to be studied, as has been demonstrated well with control experiments. The conclusion is reached, therefore, that this relatively simple device can be used routinely for the optimalization of enzyme cytochemistry of single cells.

Acrylic Resins↗
PubMed2003

Improved recovery of DNA from polyacrylamide gels after in situ DNA footprinting.

Methods used to date for the isolation of DNA from polyacrylamide gels are elution based, time-consuming and with low yield in DNA. This paper describes an improved system employing polyacrylamide gels made of a meltable matrix. The new system was successfully applied to in situ DNA footprinting following gel retardation assays.

Acrylic Resins↗
PubMed1988

Steric protection of a modified glass surface with adsorbed polyacrylamide: interactions with blood proteins.

Glass beads chemically modified with aluminol (A1OH) groups were coated with a monolayer of high-molecular-mass polyacrylamide at different temperatures and exposed to 125I-labelled albumin and fibrinogen solutions for 1 h. No adsorption of albumin was observed, whereas fibrinogen adsorption increased as a function of temperature. These observations were interpreted in terms of the density and stability of the adsorbed polymer layer. At 25 degrees C, where fibrinogen adsorption was already important, it was shown, using 3H-labelled polyacrylamide, that fibrinogen partly displaced the polymer from its adsorption sites.

Acrylic Resins↗
PubMed1993

Affinity electrophoresis in multisectional polyacrylamide slab gels is a useful and convenient technique for measuring binding constants of aryl sulfonamides to bovine carbonic anhydrase B.

This paper describes convenient preparations of heterogeneous multisectional polyacrylamide slab gels and the protocols that use these gels to measure protein-ligand binding constants [using bovine carbonic anhydrase B (CAB) as a model system]. Unlike procedures for affinity electrophoresis using tube gels, all binding information concerning protein-ligand interactions was encoded in a single multisectional gel: the procedure involving for measuring binding constants required no postelectrophoresis manipulation of gels. Use of these types of gels improves the accuracy of affinity gel electrophoresis (AGE) by providing reliable internal protein standards. Binding constants measured by AGE agree with those determined in homogeneous solution by spectrophotometric measurements. This technique has been used to investigate the influence of the length of the spacer separating the ligand and the polyacrylamide backbone on the binding constants. Dissociation constants obtained using the affinity gels approach the values measured in free solution, when the spacer is sufficiently long (> or = 18 A); affinity ligands having short spacers give high apparent dissociation constants.

Animals↗
PubMed2006

Integrated preconcentration SDS-PAGE of proteins in microchips using photopatterned cross-linked polyacrylamide gels.

The potential of integration of functions in microfluidic chips is demonstrated by implementing on-chip preconcentration of proteins prior to on-chip protein sizing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two polymeric elements-a thin (approximately 50 microm) size exclusion membrane for preconcentration and a longer (approximately cm) porous monolith for protein sizing-were fabricated in situ using photopolymerization. Contiguous placement of the two polymeric elements in the channels of a microchip enabled simple and zero dead volume integration of the preconcentration with SDS-PAGE. The size exclusion membrane was polymerized in the injection channel using a shaped laser beam, and the sizing monolith was cast by photolithography using a mask and UV lamp. Proteins injected electrophoretically were trapped on the upstream side of the size exclusion membrane (MW cutoff approximately 10 kDa) and eluted off the membrane by reversing the electric field. Subsequently, the concentrated proteins were separated in a cross-linked polyacrylamide monolith that was patterned contiguous to the size exclusion membrane. The extent of protein preconcentration is easily tuned by varying the voltage during injection or by controlling the sample volume loaded. Electric fields applied across the nanoporous membrane resulted in a concentration polarization effect evidenced by decreasing current over time and irreproducible migration of proteins during sizing. To minimize the concentration polarization effect, sieving gels were polymerized only on the separation side of the membrane, and an alternate electrical current path was employed, bypassing the membrane, for most of the elution and separation steps. Electrophoretically sweeping a fixed sample volume against the membrane yields preconcentration factors that are independent of protein mobility. The volume sweeping method also avoids biased protein loading from concentration polarization and sample matrix variations. Mobilities of the concentrated proteins were log-linear with respect to molecular weight, demonstrating the suitability of this approach for protein sizing. Proteins were concentrated rapidly (<5 min) over 1000-fold followed by high-resolution separation in the sieving monolith. Proteins with concentrations as low as 50 fM were detectable with 30 min of preconcentration time. The integrated preconcentration-sizing approach facilitates analysis of low-abundant proteins that cannot be otherwise detected. Moreover, the integrated preconcentration-analysis approach employing in situ formation of photopatterned polymeric elements provides a generic, inexpensive, and versatile method to integrate functions at chip level and can be extended to lowering of detection limits for other applications such as DNA analysis and clinical diagnostics.

Acrylic Resins↗
PubMed1983

Anomalous electrophoresis of deoxyribonucleic acid restriction fragments on polyacrylamide gels.

A detailed study has been made of the polyacrylamide gel electrophoresis of DNA restriction fragments obtained from two plasmids, pBR322 and p82-6B. Variables studied were molecular weight, gel concentration, temperature, and electric field strength. The retardation coefficients of the larger fragments (greater than 800 base pairs) were independent of molecular weight. The retardation coefficients of the smallest fragments (less than or equal to 300 base pairs) were proportional to Mr1/3, and therefore to the mean geometric radii of the fragments. The logarithm of the relative mobility of all fragments was also proportional to Mr1/3. The anomalous migration of certain fragments on polyacrylamide gels was found to be "transportable" into fragments generated by different restriction enzymes. Anomalous migration was enhanced at lower temperatures and disappeared upon increasing the temperature. A fragment which migrated anomalously slowly migrated even more anomalously when dimerized; dimerizing a normally migrating fragment resulted in the normal migration of the dimerized fragment. Anomalously migrating fragments were found to be localized in distinct regions of the pBR322 circle.

DNA↗
PubMed1988

Covalent immobilization of proteins for high-sensitivity sequence analysis: electroblotting onto chemically activated glass from sodium dodecyl sulfate-polyacrylamide gels.

We report a new method for the preparation of proteins in a form suitable for high-sensitivity N-terminal amino acid sequence analysis. Proteins separated by polyacrylamide gel electrophoresis were electrophoretically transferred onto glass fiber filter paper chemically activated by the introduction of phenyl isothiocyanate functional groups. The proteins became covalently coupled to the matrix during the electrotransfer process. Bands containing transferred proteins were detected by fluorescent staining or autoradiography, cut out from the glass fiber filter, and directly loaded into the cartridge of a gas-phase sequenator. The covalent nature of the interactions between protein and glass fiber support permitted the use of more vigorous solid-phase sequencing protocols and of alternative sequencing reagents. This high-efficiency isolation and covalent coupling method provides the essential first step toward enhanced-sensitivity protein sequence analysis. The method has been successfully applied to the isolation of a wide variety of proteins from SDS-polyacrylamide gels, and was shown to be compatible with both the standard Edman reagent phenyl isothiocyanate and alternative sequencing reagents such as 4-(N,N'-dimethylamino)azobenzene-4'-isothiocyanate (DABITC).

Amino Acid Sequence↗
PubMed1975

Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.

We describe the use of polyacrylamide gel electrophoresis to estimate chain lengths of double- and single-stranded DNA molecules in the size range 20-1000 base pairs (or nucleotides). Double-stranded DNA molecules of known length produced either by organic synthesis or by restriction endonuclease digestion of viral DNAs were used as standards. The relative electrophoretic mobilities of these standards were examined on both nondenaturing (aqueous) polyacrylamide gels and on denaturing gels containing 7 M urea or 98% formamide. Electrophoretic mobility of DNA is a linear function of the log of molecular weight if appropriate conditions are used, although exceptions are noted. Chain lengths can be conveniently estimated by using as standards bacteriophage gamma DNA restriction fragments or commercially available tracking dyes.

Base Sequence↗
PubMed2005

Efficient analysis of egg yolk proteins and their thermal sensitivity using sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing and nonreducing conditions.

The multiple functional properties of egg yolk are mostly influenced by its complex protein composition. The high lipid content of egg yolk as well as the low solubility of delipidated egg yolk lipoproteins make analysis by conventional chromatographic or electrophoretic techniques a difficult task. This work describes a method to profile egg yolk proteins after delipidation with acetone using sodium dodecyl sulfate polyacrylamide gel electrophoresis on precast 8-18% T polyacrylamide gradient gels. Twenty bands were obtained for the whole egg yolk profile with molecular weights ranging between 5 and 221 kDa. The bands were identified based on their molecular weight and by comparison with isolated egg yolk subfractions. The dissociation behavior under reducing and nonreducing conditions provided additionally helpful information for identification and characterization of the yolk proteins. The method presented is very well suited for assaying the thermal sensitivity of whole yolk and its components and thus for the characterization of heat treatment processes.

Acetone↗
PubMed2006

Microwave-assisted one-step synthesis of polyacrylamide-metal (M = Ag, Pt, Cu) nanocomposites in ethylene glycol.

Polyacrylamide-metal (M = Pt, Ag, Cu) nanocomposites with metal nanoparticles homogeneously dispersed in the polymer matrix have been successfully prepared with the corresponding metal salt and acrylamide monomer in ethylene glycol by microwave heating. This method is based on the single-step simultaneous formation of metal nanoparticles and polymerization of the acrylamide monomer, leading to a homogeneous distribution of metal nanoparticles in the polyacrylamide matrix. Ethylene glycol acts as both a reducing reagent and a solvent, thus no additional reductant is needed. Another advantage is that no initiator for AM polymerization and no surfactant for stabilization of metal nanoparticles are necessary. The products were characterized by X-ray powder diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FTIR), ultraviolet visible (UV-vis) absorption spectra, and thermogravimetric (TG) and differential scanning calorimetric analysis (DSC).

Acrylic Resins↗
PubMedDate not supplied

Direct atmospheric pressure coupling of polyacrylamide gel electrophoresis to mass spectrometry for rapid protein sequence analysis.

Using laser desorption-atmospheric pressure chemical ionization we describe a novel approach for coupling mass spectrometry to polyacrylamide gel electrophoresis. In contrast to other approaches, the method allows for the direct sampling of a polyacrylamide gel-embedded protein without the addition of any exogenous matrixes and is performed at atmospheric pressure. After electrophoresis and enzymatic digestion, the gel is analyzed at AP by photons that desorb neutral peptide molecules, followed by corona discharge ionization in the gas-phase, and subsequent mass analysis. Our experimental results demonstrate the method to (1) rapidly identify electrophoresed proteins via "peptide fingerprinting" using protein databases, (2) detect single-amino acid polymorphisms, and (3) has potential for sub-picomole sensitivity while still maintaining in situ gel desorption-ionization at ambient conditions.

Atmospheric Pressure↗
PubMed2006

VDAC1, having a shorter N-terminus than VDAC2 but showing the same migration in an SDS-polyacrylamide gel, is the predominant form expressed in mitochondria of various tissues.

The voltage-dependent anion channel (VDAC) is a pore-forming protein expressed in the outer membrane of eukaryotic mitochondria. Three isoforms of it, i.e., VDAC1, VDAC2, and VDAC3, are known to be expressed in mammals; however, the question as to which is the main isoform in mitochondria is still unanswered. To address this question, we first prepared standard VDACs by using a bacterial expression system and raised various antibodies against them by using synthetic peptides as immunogens. Of the three bacterially expressed VDAC isoforms, VDAC3 showed faster migration in SDS-polyacrylamide gels than VDAC1 and VDAC2, although VDAC2 is longer than VDAC1 and VDAC3, due to a 12-amino acid extension of its N-terminal region. Even with careful structural characterization of the expressed VDACs by LC-MS/MS analysis, serious structural modifications of VDACs causing changes in their migration in SDS-polyacrylamide gels were not detected. Next, immunoreactivities of the raised antibodies toward these bacterially expressed VDAC isoforms were evaluated. Trials to prepare specific antibodies against the three individual VDAC isoforms were not successful except in the case of VDAC1. However, using a synthetic peptide corresponding to the highly conserved region among the three VDACs, we were successful in preparing an antibody showing essentially equal immunoreactivities toward all three VDACs. When mitochondrial outer membrane proteins of various rat tissues were subjected to 2-dimensional electrophoresis followed by immunoblotting with this antibody, six immunoreactive protein spots were detected. These spots were characterized by LC-MS/MS analysis, and the signal intensities among the spots were compared. As a result, the signal intensity of the spot representing VDAC1 was the highest, and thus, VDAC1 was concluded to be the most abundantly expressed of the three VDAC isoforms in mammalian mitochondria.

Amino Acid Sequence↗
PubMed1998

Comparison of biochemical effects produced by calcium ions and by monomers of polyacrylamide (acrylamide and bisacrylamide) on strains of Saccharomyces cerevisiae used for production of chiral synthons.

The biochemical behaviour of four commercial strains of Saccharomyces cerevisiae was studied in the presence of calcium ions, acrylamide and bisacrylamide. Calcium ions at a concentration of 300 microM induced an increase of NAD(P)+ reduction in commercial Turkish and American strains, while in Chilean and Brazilian commercial strains, it diminished NAD(P)+ reduction. On the other hand, polyacrylamide monomers (acrylamide and bisacrylamide) induced a decrease of NAD(P)+ reduction in all strains studied in this paper. When membrane potential (delta psi) and oxygen consumption were measured in the presence of polyacrylamide monomers, a decrease of both was observed in all strains studied.

Acrylamides↗