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PubMed2005

Quasi-interpenetrating network formed by polyacrylamide and poly(N,N-dimethylacrylamide) used in high-performance DNA sequencing analysis by capillary electrophoresis.

Quasi-interpenetrating network (IPN) formed by polyacrylamide and poly(N,N-dimethylacrylamide) was designed, synthesized, and tested as a high-performance DNA separation medium by capillary electrophoresis. The performance of quasi-IPN on DNA sequencing was determined by the acrylamide to dimethylacrylamide molar ratio, polyacrylamide molecular weight, and its size distribution. Under optimal operating conditions, quasi-IPN was able to achieve one-color DNA sequencing up to 1000 bases in 39 min, or 1200 bases in 60 min. Its performance was compared with some of the existing commercialized products, such as POP6 from Applied Biosystems and MegaBACE matrix from Amersham Biosciences. By using the ABI 310 Genetic Analyzer, even without optimized base-calling software, quasi-IPN yielded a read length of up to 700 bases of contiguous sequence (50-750 bases) in 35 min with 99.6% accuracy, or 750 bases of contiguous sequence (50-800 bases) in 37 min with 98.0% accuracy.

Acrylamides↗
PubMed2006

Silver staining method for DNA in polyacrylamide gels using eriochrome black T as a silver-ion sensitizer.

A sensitive silver staining method using eriochrome black T as a silver-ion sensitizer for DNA detection in polyacrylamide gels was developed. The sensitivity of this staining method was significantly improved by the new silver-ion sensitizer containing a diazo group, which has reducing power. The staining method lasted a total of approximately 15 min following a fixing step for 2 x 20 min. The detection limit of this staining method was 1-4 pg for PhiX174 DNA/HaeIII in both nondenaturing and denaturing polyacrylamide gels. This staining method was especially effective in low-base pair DNA, with a sensitivity that was approximately ten-fold higher than previously published silver staining methods.

Acrylic Resins↗
PubMed2006

Using in situ rheology to characterize the microstructure in photopolymerized polyacrylamide gels for DNA electrophoresis.

Photopolymerized cross-linked polyacrylamide hydrogels are attractive sieving matrix formulations for DNA electrophoresis owing to their rapid polymerization times and the potential to locally tailor the gel pore structure through spatial variation of illumination intensity. This capability is especially important in microfluidic systems, where photopolymerization allows gel matrices to be precisely positioned within complex microchannel networks. Separation performance is also directly related to the nanoscale gel pore structure, which is in turn strongly influenced by polymerization kinetics. Unfortunately, detailed studies of the interplay among polymerization kinetics, mechanical properties, and structural morphology are lacking in photopolymerized hydrogel systems. In this paper, we address this issue by performing a series of in situ dynamic small-amplitude oscillatory shear measurements during photopolymerization of cross-linked polyacrylamide electrophoresis gels to investigate the relationship between rheology and parameters associated with the gelation environment including UV intensity, monomer and cross-linker composition, and reaction temperature. In general, we find that the storage modulus G' increases with increasing initial monomer concentration, cross-linker concentration, and polymerization temperature. The steady-state value of G', however, exhibits a more complex dependence on UV intensity that varies with gel concentration. A simple model based on rubber elasticity theory is used to obtain estimates of the average gel pore size that are in surprisingly good agreement with corresponding data obtained from analysis of DNA electrophoretic mobility in gels cast under identical polymerization conditions.

Acrylic Resins↗
PubMed2006

A robust cross-linked polyacrylamide coating for microchip electrophoresis of dsDNA fragments.

Surface derivatization plays an important role in microchip electrophoresis. It not only enhances the resolution, but also improves the reproducibility. So far, the most popularly used derivatization method for glass microchannels is to covalently attach a layer of linear polyacrylamide (LPA) to the channel surfaces. However, LPA coating has two problems: incomplete coverage and limited lifetime. To address these issues, we have recently developed a cross-linked polyacrylamide (CPA) derivatization protocol and demonstrated it for high-resolution protein separations by CIEF, CGE, and CZE. In this report, we used this protocol to coat microchip channels and exhibited the reliability and robustness of CPA coating for microchip electrophoresis of DNA molecules. dsDNA fragments were used as our test samples. High resolutions were obtained for fragments ranging from 100 bp to 10 kpb. After more than 800 runs, the CPA-coated microchannels still performed well and comparable resolutions were maintained throughout these runs.

Acrylic Resins↗
PubMed2002

In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide bridged, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF-pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O-glycosylated proteins up to 150 kDa after SDS-PAGE separation.

Animals↗
PubMed2002

Laser desorption-atmospheric pressure chemical ionization: a novel ion source for the direct coupling of polyacrylamide gel electrophoresis to mass spectrometry.

Laser desorption-atmospheric pressure chemical ionization-mass spectrometry (LD-APCI-MS) is presented for the atmospheric pressure (AP) sampling of tryptic peptides directly from a polyacrylamide gel. In contrast to other gel sampling mass spectrometric approaches, this technique does not require the addition of any exogenous matrices to the gel to assist with ionization. In this arrangement, a CO(2) laser at 10.6 micro m is used to desorb intact neutral peptide molecules from the gel, followed by ionization in the gas-phase with APCI. The ions are then sampled via a heated capillary inlet and transferred to a quadrupole ion trap mass spectrometer for mass analysis. Preliminary results suggest the polyacrylamide gel electrophoresis-LD-APCI-MS technique provides several advantages that could translate into a more convenient, robust methodology for the rapid identification and characterization of proteins. Finally, strategies regarding the further development of the method are presented.

Amino Acid Sequence↗
PubMed1981

Prolongation of effect of asparaginase by implantation in polyacrylamide in rats.

Asparaginase was immobilized in spherical polyacrylamide microparticles, which were inserted in polyacrylamide gel for implantation in rats. Intraperitoneal implantation of this gel depressed systemic L-asparagine for the same duration as was achieved with microparticles, i.e., 8-10 days. The duration of the effect was prolonged up to about 25 days when the gel with the enzyme was implanted subcutaneously on the back of the rat. In both cases, a collagenous capsule formed around the gel, eventually preventing contact with the circulation. The effect of a second implantation was of the same magnitude as the first one.

Acrylic Resins↗
PubMed1993

Combined alcian blue and silver staining of subnanogram quantities of proteoglycans and glycosaminoglycans in sodium dodecyl sulfate-polyacrylamide gels.

Proteoglycans stain weakly in polyacrylamide gels by traditional protein stains such as coomassie brilliant blue or silver. In the present work preparations of large aggregating proteoglycan from human articular cartilage were used to evaluate a convenient staining method based on successive staining with alcian blue and neutral silver. The method is developed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis; with ultrathin minigels in a semiautomated electrophoresis system, takes 1 1/2 h, and uses stable reagents. Preparations, electrophoresis, and staining of up to 24 samples can be completed within 2 1/2 h. The method has a detection limit of 0.04-1 ng proteoglycan and less than 0.5 ng of glycosaminoglycan. In addition the method can be adjusted for selective staining of proteoglycans and glycosaminoglycans.

Acrylic Resins↗
PubMed1993

Detection of proteins on polyacrylamide gels using calconcarboxylic acid.

We describe here a protein staining method in polyacrylamide gels with a new staining dye, 1-(2-hydroxy-4-sulfo-1-naphthylazo)-2-hydroxy-3-naphthoic acid (calconcarboxylic acid, NN). This method can be performed by both simultaneous and postelectrophoretic staining techniques. Simultaneous staining using 0.01% of NN in upper reservoir buffer eliminates the poststaining step, and thus enables detection of the proteins more rapidly and simply. In poststaining, proteins can be stained by a 30-min incubation of a polyacrylamide gel in 40% methanol/7% acetic acid solution of 0.05% NN. These techniques produced protein staining patterns identical to the ones obtained by the conventional poststaining with Coomassie blue R-250 (CB). NN staining can detect as little as 10 ng of bovine serum albumin by poststaining and 25 ng by simultaneous staining, compared to 50 ng detectable by CB poststaining. In comparing the relationship between band intensity and amount of protein, NN staining gave better linearity than CB staining.

Azo Compounds↗
PubMed1993

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis for M(r) estimations of high-molecular-weight polypeptides.

A convenient method was established for the M(r) estimation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of large polypeptides in the range from 250,000 to 600,000. Such polypeptides had previously been shown to migrate improperly in various gel electrophoresis systems. In a conventional Laemmli system with 3% polyacrylamide, approximately linear log M(r) vs Rf plots could be obtained by varying the gel thickness which ranged from 0.4 to 1.5 mm. In the additional presence of an electrolyte, such as NaCl, running times were shorter and for evaluation linear plots could be constructed from runs in very thin gels with less than 0.5 mm thickness. Handling of gels was greatly facilitated by fixing them to one of the gel chamber glass plates via bind-silane treatment prior to casting.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Analysis of recombinant Schistosoma mansoni antigen rSmp28 by on-line liquid chromatography-mass spectrometry combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis.

A recombinant Schistosoma mansoni antigen produced in Saccharomyces cerevisiae and purified by glutathione-Sepharose affinity chromatography was analyzed by tryptic peptide mapping using on-line reversed-phase high-performance liquid chromatography pneumatically assisted electrospray mass spectrometry confirming the complete primary structure. Partial covalent modification of the single cysteine in the protein with glutathione as well as partial dimerization of the Cys-containing tryptic peptide was observed. Combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and tryptic digestion of the monomeric protein in the gel slice revealed that dimerization was occurring during enzymatic digestion. Furthermore, part of the Cys-containing fragment was covalently modified with one moiety of beta-mercaptoethanol by the electrophoresis sample buffer and five of the seven methionine-containing peptide fragments were partially oxidized to the respective sulfoxides. The use of capillary columns provided a complete peptide map of rSmp28 on 7 pmol of tryptic digest after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Amino Acid Sequence↗
PubMed1994

Colorimetric detection of DNA polymerase activity after sodium dodecyl sulfate polyacrylamide gel electrophoresis.

A nonradioactive method is developed to detect DNA polymerase activity after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis containing gapped DNA as template. The technique is based on the use of digoxigenin- or biotin-labeled deoxynucleotides during DNA synthesis, and their detection by means of an anti-digoxigenin antibody-alkaline phosphatase conjugate or by a streptavidin-alkaline phosphatase conjugate. The detection of the DNA polymerase catalytic subunit is achieved after incubation of the gels with colorimetric alkaline phosphatase substrates. The technique is able to detect nanogram amounts of Escherichia coli DNA polymerase I and picogram amounts of its Klenow fragment. The results with other DNA polymerases and E. coli extracts suggest that this colorimetric detection system could be used for the analysis of an extended range of DNA polymerase enzymes. The method presented in this report offers an alternative to the already described radioactive techniques for detection of DNA polymerase activity after SDS-polyacrylamide gel electrophoresis.

Colorimetry↗
PubMed1995

PhosphorImager enhancement of sedimentation equilibrium-quantitative polyacrylamide gel electrophoresis: a highly sensitive technique for quantitation of equilibrium gradients of individual components in mixtures.

The technique called sedimentation equilibrium-quantitative polyacrylamide gel electrophoresis (Darawshe et al. (1993) Anal. Biochem. 215, 236-242) has been extended to permit the quantitation and analysis of gradients of individual radiolabeled components in a mixture of radiolabeled solutes centrifuged to sedimentation equilibrium. Immediately following centrifugation, the contents of a sample tube are fractionated into aliquots corresponding to laminae of solution at different radial positions in the centrifuge. Following treatment with sodium dodecyl sulfate-containing buffer, a portion of each fraction is subjected to electrophoresis on a polyacrylamide gel. The gel is then incubated with a strong phosphor plate and subsequently scanned with a Molecular Dynamics PhosphorImage. The concentration of an individual radiolabeled component at a particular radial distance is proportional to the integrated intensity of the image of the radiolabeled band of that component in the fraction corresponding to that radial distance. Concentration gradients reconstructed in this fashion are interpreted in the context of conventional sedimentation equilibrium theory. The results of control experiments carried out with purified proteins of known molar mass and the measurement of the molar mass of a new, partially purified protein are reported.

Animals↗
PubMed1996

Separation by blue native and colorless native polyacrylamide gel electrophoresis of the oxidative phosphorylation complexes of yeast mitochondria solubilized by different detergents: specific staining of the different complexes.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) or colorless native polyacrylamide gel electrophoresis (CN-PAGE) allowed separation of the oxidative phosphorylation complexes of yeast mitochondria. These complexes were characterized by specific staining related to their enzymatic activity. Solubilization of mitochondria by different nonionic detergents such as Triton X-100, dodecyl maltoside, Nonidet P-40, Lubrol, octyl glucoside, or Hecameg led to the separation of F1-FO ATPase complexes exhibiting distinct apparent molecular masses related to different contaminating proteins and lipids. All these different forms were active in ATP hydrolysis as revealed directly on the gel. Analysis of the subunit composition of these complexes was carried out by a two-dimensional Tricine-SDS-PAGE and showed that the purest F1-FO ATPase complex was obtained with Lubrol, whereas with Hecameg and octyl glucoside, only the F1 part of ATPase was solubilized.

Chemistry Techniques, Analytical↗
PubMed1997

Sensitive reverse staining of bacterial lipopolysaccharides on polyacrylamide gels by using zinc and imidazole salts.

We present a new method for visualizing bacterial lipopolysaccharides (LPS)/lipooligosaccharides (LOS) electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, gels are washed in boiling water to appreciably remove remaining electrophoresis reagents, then incubated in 10 mM zinc sulfate for 15 min, and subsequently immersed in 0.2 M imidazole for 3 min. As a result, zinc salts precipitate all over the gel surface except in the zones occupied by LPS/LOS, which appear as transparent, colorless bands. Gels can be stored in distilled water for weeks without loss of the negative image. The sensitivity of this stain is similar to that of silver. We believe that zinc-imidazole may be a suitable nontoxic alternative to silver in the rapid analysis of LPS/LOS by polyacrylamide gel electrophoresis.

Coloring Agents↗
PubMed1997

Detection of protein phosphatase activities in sodium dodecyl sulfate-polyacrylamide gel using peptide substrates.

A method for detection of protein phosphatase activity toward phosphorylated oligopeptides in SDS-polyacrylamide gel was developed. A synthetic peptide (MHRQETVDC) corresponding to the autophosphorylation site of calmodulin-dependent protein kinase II (residue 281-289) was conjugated to poly-L-lysine and phosphorylated with [gamma-32P]ATP by the action of calmodulin-dependent protein kinase II, and the [32P]-phosphopeptide-polymer conjugate was included as a substrate for protein phosphatases in gels. When a crude extract from rat brain was electrophoresed on polyacrylamide gel containing the [32P]phosphopeptide conjugate, followed by treatment for in situ renaturation and autoradiography, three transparent bands corresponding to apparent molecular weights of 52,000, 58,000 and 74,000, resulting from the removal of the [32P]phosphate from the phosphopeptide conjugate included in the gel were observed, indicating the existence of at least three different phosphoprotein phosphatases catalyzing dephosphorylation of the phosphopeptide in the brain. Among the three, two bands corresponding to molecular weights of 52,000 and 58,000 were not clearly observed when other phosphopeptide-polymer conjugates such as C-syntide-2 and CAMKAKS peptide were included in gels, suggesting that site-specific protein phosphatases can be detected in crude tissue extracts by this in-gel protein phosphatase assay.

Amino Acid Sequence↗
PubMed2002

Identification of polysaccharide binding proteins by affinity electrophoresis in inhomogeneous polyacrylamide gels and subsequent SDS-PAGE/matrix-assisted laser desorption ionization-time of flight analysis.

A procedure that allows the identification of polysaccharide binding polypeptides is described. The method can be applied to proteins whose enzymatic activity is either unknown or cannot be identified unambiguously by activity-staining procedures and it has been used for very complex protein mixtures, such as crude extracts of plant organs. The procedure consists of three steps. First, an affinity polyacrylamide gel electrophoresis using an inhomogeneous polyacrylamide slab gel composed of two triangular parts, an upper gel lacking the ligand and a lower triangular gel containing an immobilized ligand, is performed. Proteins that interact with the ligand form bands that deviate from those of nonbinding proteins and can be detected by protein staining (or, if possible, by activity staining). Second, the bands containing the interacting proteins are excised, denatured, and subjected to SDS-PAGE using a slab gel. In the resulting protein pattern the target proteins cover most of the length of the gel piece applied to the SDS gel, whereas contaminating proteins appear as spots or narrow bands. Suitable regions of the target protein bands are selected for tryptic digestion. Third, the resulting peptides are analyzed by matrix-assisted laser desorption ionization-mass spectrometry followed by database research.

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

A gel-concentration-independent retardation detected in two fragments of the rrnB P1 promoter of E. coli using transverse polyacrylamide pore gradient gel electrophoresis.

Two fragments of the E. coli rrnB ribosomal RNA P1 promoter Upstream Activation Region exhibit a constant gel retardation, over a polyacrylamide gel concentration range of 3% to 10%. Gel retardation is usually seen to increase with polyacrylamide gel concentration as in the case of the 219 base pair Crithidia fasciculata kinetoplast DNA fragment which represents the classic case of sequence directed curvature. Computer modeling of the Upstream Activation Region fragments suggests that their unusual electrophoretic behavior can be accounted for on the basis of a "screw" model proposed by Drak and Crothers (1991, Proc. Natl. Acad. Sci. USA 88,3074-3078).

Animals↗