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PubMed1991

Gel electrophoresis in the presence of soluble, aqueous polymers: horizontal sodium dodecyl sulfate-polyacrylamide gels.

Because little is known about the use of aqueous polymers in polyacrylamide gel electrophoresis, we undertook a feasibility study that enables the discontinuous Laemmli-formulated system of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis to be performed in a horizontal format by the addition of large-sized aqueous polymers (i.e., dextrans and methylcelluloses). We studied four parameters: the cross-linking agent (bisacrylamide vs AcrylAide) and the polymer concentration, nature, and size. Three concentrations of each polymer were used. The best differentiation between the standard markers and the sharpest bands were obtained using concentrations of 2.5 and 0.06% (w/v) for Dextran T-500 and methylcellulose 4000, respectively. There was no predictable pattern to the variation in the plots of log Mr vs Rf caused by varying the concentration and length of the dextrans; however, the methylcellulose patterns suggest that gel viscosity is important. The results suggest that the combination of 0.06% methylcellulose 4000 polymers with bisacrylamide is a convenient and inexpensive means of performing flatbed Laemmli SDS-polyacrylamide gel electrophoresis.

Dextrans↗

PubMed1991

Eosin Y staining of proteins in polyacrylamide gels.

A staining method is described in which various proteins in polyacrylamide gels can be stained by using eosin Y. After a brief incubation of a polyacrylamide gel in an acidic solution of 1% eosin Y, various proteins, including human erythrocyte membrane sialoglycoproteins which are not detectable by Coomassie blue R-250 (CB), can be detected with a sensitivity of 10 ng protein. This is far more sensitive than CB staining and is comparable to the sensitivity of silver staining. In a Western blot, the antigenicity of an eosin Y stained protein is retained. In addition, proteins on an immunoblot sheet can be detected by eosin Y staining. The method described is rapid, sensitive, and reproducible with various proteins in polyacrylamide gels and has the added advantage of also staining sialoglycoproteins.

Acrylic Resins↗

PubMed1976

The electrophoretic mobility of double-stranded RNA in polyacrylamide gels as a function of molecular weight.

A re-evaluation of the mobility of double-stranded RNA on polyacrylamide gels over a molecular weight range of 0.46-6.3 . 10(6) was carried out using double-stranded RNAs of: bacteriophage ø6; virus like particles or mycoviruses of Penicillium chyrsogenum, Penicillium stoloniferum and Helminthosporium maydis, and reovirus type III. When the relative mobility on polyacrylamide gels was plotted as a function of log molecular weight, a smooth curve could be drawn which passed through all points. The implications of these findings to the determination of molecular weight of double-stranded RNA by polyacrylamide gel electrophoresis are discussed.

Binding Sites↗

PubMed1978

Purification of RNAase II by preparative polyacrylamide gel electrophoresis.

Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K+ and Mg2+ and hydrolyses poly(A) to 5'-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913--922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 +/- 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 +/- 2000, 33 000 +/- 2000, and 26 000 +/- 1000. The enzyme thus appears to consist of three dissimilar subunits.

Chromatography, DEAE-Cellulose↗

PubMed1975

Electrofocusing followed by gradient electrophoresis: a two-dimensional polyacrylamide gel technique for the separation of proteins and its application to the immunoglobulins.

A two-dimensional polyacrylamide gel technique for the separation of proteins is described. Electrofocusing in a 3.5% polyacrylamide gel is followed by electrophoresis through a 4-24% concave polyacrylamide gel gradient. The method resolves a large number of serum proteins and produces highly reproducible patterns. It is particularly useful for studies involving proteins of high molecular weight, such as alpha2-macroglobulin, beta-lipoprotein and IgM. The application of the technique to the immunoglobulins in pathological sera is reported.

Blood Proteins↗

PubMed1982

Localization of gamma-glutamyl transferase on polyacrylamide gels using L-gamma-glutamyl-p-nitroanilide as substrate.

A method has been developed for the localization of gamma-glutamyltransferase (gamma-GT) on polyacrylamide gels using L-gamma-glutamyl-p-nitroanilide as substrate. The optimum conditions for staining on polyacrylamide gels with this substrate were evaluated and compared with the staining method using N-gamma-L-glutamyl-alpha-naphthylamide as substrate. Using papain digested, purified gamma-GT from human fetal liver it was shown that the method could be used for quantitation of the enzyme on polyacrylamide gels. It was also shown that identical patterns of staining for serum gamma-GT could be obtained with L-gamma-glutamyl-p-nitroanilide and N-gamma-L-glutamyl-alpha-naphthylamide as substrate.

Acrylic Resins↗

PubMed1993

Separation of DNA fragments by capillary electrophoresis using replaceable linear polyacrylamide matrices.

The use of low percent (1.5-6% T) replaceable linear polyacrylamide (LPA) network matrices for rapid separation of double-stranded DNA fragments was explored. Separations of fragments ranging from 20 to 23,000 base pairs were readily achieved. Typically, 4 x 10(6) theoretical plates/m were obtained in less than 30 min. Short separation times under 2 min were also possible, using the DNA intercalating dye, ethidium bromide, along with high electric fields. The high resolving power of linear polyacrylamide was demonstrated in the separation of two fragments which differ by a single base pair (123/124 base pairs) using 6% T LPA and ethidium bromide intercalation. This LPA composition allowed for the possible single base-pair resolution of dsDNA fragments up to 300 base pairs in length. Several concentrations of the linear polyacrylamide for different ranges of fragment lengths have been employed. In addition, replaceable LPA offers the advantage of a fresh separation matrix for each run, thus overcoming column stability problems and minimizing needs for sample cleanup. Electro-osmotic flow was substantially reduced using stable capillary coatings, which were required for obtaining high efficiencies and good reproducibility.

Bacteriophage phi X 174↗

PubMed1984

Counterimmunoblotting: detection of non-denatured or denatured antigens in antibody-containing agarose gels following polyacrylamide gel electrophoresis.

Utilizing the principles of counterimmunoelectrophoresis, a technique was devised to immunologically identify non-denatured protein antigens resolved by polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred from polyacrylamide gel to antibody-containing agarose gel in a commercially available blotting apparatus. Detection of the antigen/antibody complex by Coomassie blue staining (in the ng range for unlabeled antigen) or autoradiography (in the pg range for radiolabeled antigen) established the sensitivity and specificity of this method. Similar sensitivity could be obtained following conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis after partial removal of the detergent and possible renaturation of proteins. Placement of a nitrocellulose sheet on the anodal side of the agarose gel produced a 'negative replica' (proteins/polypeptides not reacting with specific antibodies in the agarose gel). Counterimmunoblotting provides an additional method for molecular weight determination of protein antigens with sensitivity and specificity comparable to established nitrocellulose blotting (unlabeled proteins) or immunoprecipitation (labeled proteins) procedures.

Antigen-Antibody Complex↗

PubMed1985

Chemiluminescence monitoring of phagocyte oxidative metabolism in mice bearing polyacrylamide induced granulomas.

A technical protocol was recently described by Fauve et al. (J. Immunol. Methods 1983, 64, 345) for inducing subcutaneous granuloma with polyacrylamide microbeads. The present study using this technique demonstrates that the capacity of host phagocytes to generate reactive oxygen species can be easily monitored by chemiluminescence, both locally in granuloma infiltrating cells and at sites remote from the inflammatory reaction, i.e., within microamounts of whole blood and in spleen cells. We observed that both resting and stimulated (zymosan or phorbol-myristate acetate) production by C57BL/6 mouse phagocytes are significantly higher in granulomas induced with high porosity polyacrylamide beads (P300) than in those induced with beads of low polyacrylamide porosity (P4). Since this selective modulation of phagocyte oxidative metabolism is also detectable within microamounts of whole blood and in spleen cells, it could serve as a model for investigating the role of reactive oxygen species in the inflammatory reaction.

Acrylic Resins↗

PubMed1989

Decreased poststenotic flow disturbance during drag reduction by polyacrylamide infusion without increased aortic blood flow.

The infusion of polyacrylamide in open chest rats has been reported to increase aortic blood flow and the effect has been ascribed to the "drag reduction" properties of these compounds. In six anesthetized dogs the infusion of polyacrylamide to a total dose of 2 mg/kg caused a reduction in midline and separation zone Doppler spectral broadening in the common carotid artery poststenotic velocity field. This apparent reduction in poststenotic turbulence was interpreted as indicating the presence of a drag reducing effect. Despite this demonstration that polyacrylamide was present in the blood in drag reducing concentrations no increase in aortic blood flow was produced.

Acrylic Resins↗

PubMed1981

A highly sensitive technique for staining DNA and RNA in polyacrylamide gels using silver.

A technique is described for staining DNA in polyacrylamide gels with silver. It is rapid, requiring about 30 min for whole staining and development procedure, very simple and at least 20 times more sensitive than ethidium bromide for the staining of double-stranded DNA in polyacrylamide gels. This technique can also be applied for the staining of denatured, single-stranded DNA as well as RNA after their electrophoretic separation on polyacrylamide gels, having the same sensitivity as for double-stranded DNA fragments.

Bacteriophage lambda↗

PubMed1984

Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose.

A simple, horizontal device for rapid electrophoretic transfer of proteins from several polyacrylamide gels simultaneously is described. Up to six 'TRANS-UNITS' consisting of soaked filter paper on either side of polyacrylamide gel/nitrocellulose sheets that are separated by dialysis membranes are stacked between graphite plate electrodes. The only buffer reservoir in the apparatus is that in stacked, soaked filter paper. A special buffer system based on the isotachophoresis theory was developed for this purpose. The electrophoretic transfer was performed with equal efficiency in all TRANS-UNITS of the stack. Only traces of a few proteins remained in the polyacrylamide gel after transfer. With this apparatus, 50 protein bands from a human serum protein sample (diluted 1 : 100) were detected by immunoblotting with the retainment of the high resolution of the SDS-PAGE technique. The apparatus provided a constant current density of 0.8 mA/cm2 during the 1-h transfer time at 21 degrees C, irrespective of the number of TRANS-UNITS. The apparatus generated 1-5 W in joule heat, depending on the number of TRANS-UNITS in the stack.

Blood Protein Electrophoresis↗

PubMed1988

Polyacrylamide concentration gradient gel electrophoresis of the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor in rat liver cytosol.

An assay based on polyacrylamide concentration gradient gel electrophoresis for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor in rat liver cytosol was developed. [3H]TCDD-labeled cytosol was treated with dextran-coated charcoal and electrophoresed for 3000 Vh in polyacrylamide concentration gradient gels (T = 2.5-20%). A Tris-borate running buffer (pH 8.35) without salt and detergents was used. When the polyacrylamide concentration gradient was concave a linear calibration curve of log molecular weight vs. migration distance was obtained for the standard proteins used. Under these conditions the Mr value of the TCDD receptor from rat liver cytosol was estimated to be 316,000 +/- 27,000 before, and 246,000 +/- 12,000 after limited proteolysis. The Bmax for the specific binding of [3H]TCDD was between 35 and 43 fmol/mg cytosolic protein and the Kd of the binding ranged from 0.8 to 1.1 nM, as judged by saturation and Scatchard analysis.

Animals↗

PubMed2005

Polyacrylamide gel electrophoresis separation and detection of polyamidoamine dendrimers possessing various cores and terminal groups.

Detection and separation of polyamidoamine dendrimers possessing various cores and surface groups was studied by polyacrylamide gel electrophoresis. Although many dyes and staining techniques were able to detect dendrimers on polyacrylamide gels, Coomassie Blue was found to be the most sensitive and convenient. Amine and hydroxyl terminated dendrimers were best separated under acidic conditions, while dendrimers with carboxyl surfaces required alkaline buffers. Some dendrimers were very susceptible to diffusion that could occur during their separation, staining or destaining steps. In the absence of an appropriate fixation step, dendrimers could be resolved by using small pore size gels and low voltage or current. Increasing core lengths did not significantly affect migration of a given dendrimer generation but exhibited improved separation and staining characteristics. Polyacrylamide gel electrophoresis was found to be a rapid, inexpensive, and reliable procedure to characterize many different water-soluble dendritic macromolecules.

Dendrimers↗

PubMed2004

Weak alignment of membrane proteins in stressed polyacrylamide gels.

Residual dipolar couplings are important as angular constraints for the structure determination of membrane proteins in micelles. Strained polyacrylamide gels are one of the few available mechanisms available for inducing the requisite weak alignment for these samples. However, their use is frequently limited by the ability to incorporate proteins and buffer solutions into the gel matrix. The implementation of several methods of incorporating membrane proteins into gels are described. Conditions for copolymerizing the protein in the absence of a change in pH are detailed. Electrophoresis is also shown to be a useful method to incorporate proteins. Weak alignment of the protein-micelle complex in the gel matrix is subsequently achieved using either vertical or radial compression. The magnitude of alignment can be controlled by altering the gel concentration, the acrylamide/bisacrylamide ratio, and the compression ratio. The alignment tensor can be altered relative to uncharged polyacrylamide gels by copolymerizing samples with acrylamide/acrylic acid to incorporate negative charges in the strained polyacrylamide gel to provide an alternate orientation.

Acrylic Resins↗

PubMed1998

Effects of supercoiling in electrophoretic trapping of circular DNA in polyacrylamide gels.

Electrophoretic velocity and orientation have been used to study the electric-field-induced trapping of supercoiled and relaxed circular DNA (2926 and 5386 bp) in polyacrylamide gels (5% T, 3.3% C) at 7.5-22.5 V/cm, using as controls linear molecules of either the same contour length or the same radius of gyration. The circle-specific trapping is reversible. From the duration of the reverse pulse needed to detrap the molecules, the average trap depth is estimated to be 90 A, which is consistent with the molecular charge and the field strengths needed to keep molecules trapped. Trapped circles exhibit a strong field alignment compared to the linear form, and there is a good correlation between the enhanced field alignment for the circles and the onset of trapping in both constant and pulsed fields. The circles do not exhibit the orientation overshoot response to a field pulse seen with linear DNA, and the rate of orientation growth scales as E(-2+/-0.1) with the field, as opposed to E(-1.1+/-0.1) for the linear form. These results show that the linear form migrates by cyclic reptation, whereas the circles most likely are trapped by impalement on gel fibers. This proposal is supported by very similar velocity and orientation behavior of circular DNA in agarose gels, where impalement has been deemed more likely because of stiffer gel fibers. The trapping efficiency is sensitive to DNA topology, as expected for impalement. In polyacrylamide the supercoiled form (superhelical density sigma = -0.05) has a two- to fourfold lower probability of trapping than the corresponding relaxed species, whereas in agarose gels the supercoiled form is not trapped at all. These results are consistent with existing data on the average holes in the plectonemic supercoiled structures and the fiber thicknesses in the two gel types. On the basis of the topology effect, it is argued that impalement during pulsed-field electrophoresis in polyacrylamide gels may be useful for the separation of more intricate DNA structures such as knots. The results also indicate that linear dichroism on field-aligned molecules can be used to measure the supercoiling angle, if relaxed DNA circles are used as controls for the global degree of orientation.

Acrylic Resins↗

PubMed1998

Rapid characterization of mutations in amplified human hprt cDNA by polyacrylamide gel electrophoresis.

Compiling hprt mutation spectra involves the isolation and analysis of numerous 6-thioguanine-resistant clones for identifying characteristic point mutations. Since cDNA amplificates are compulsory intermediates in most mutant classification protocols, we suggest their preliminary characterization by polyacrylamide gel electrophoresis for the rapid distinction of clonal and independent mutants and for streamlining mutant analysis procedures. Based on the human hprt cDNA sequence a strategy was developed for mapping missing exons by analytical digests with a small panel of restriction enzymes. In mutant classification schemes, polyacrylamide gel electrophoresis of AluI-digested cDNA amplificates increased the sensitivity for detecting RT-PCR products of reduced size, e.g., in the case of missing exon 5. Restriction analysis of cDNA amplificates from 109 independent mutant clones showed a significant increase of exon loss after NNK induction as compared to spontaneous or BaP-induced mutants. The determination of exon loss from cDNA amplificates, as carried out for 39 independent mutant PCR products, might direct towards the genomic target sequences carrying the point mutations, that caused the aberrant splicing, thus eliminating the need of laborious multiplex PCR comprising all exons. For single-strand conformation polymorphism (SSCP) analysis of five known point mutations, sub-amplificates comprising exons 7 and 8 of hprt cDNA were obtained. After a combined heat and alkali denaturation of the double-stranded PCR products, the samples were separated in pre-cast polyacrylamide gels under non-denaturing conditions. Five known nucleotide substitutions within the amplified region, including the C508T hot spot mutation, resulted in mobility shifts of single-strand bands relative to the wild type pattern.

Cell Line↗

PubMed2004

Direct from polyacrylamide gel infrared laser desorption/ionization.

The direct combination of gel electrophoresis and infrared laser desorption/ionization time-of-flight mass spectrometry has been demonstrated. We present results for infrared laser desorption and ionization mass spectrometry of peptides and proteins directly from a polyacrylamide gel without the addition of a matrix. Analyte molecules up to 6 kDa were ionized directly from a vacuum-dried sodium dodecyl sulfate-polyacrylamide gel after electrophoretic separation. Mass spectra were obtained at the wavelength of 2.94 microm, which is consistent with IR absorption by N-H and O-H stretch vibrations of water and other constituents of the gel. A 5-nmol quantity of peptide or protein was loaded per gel slot, although it was possible to obtain mass spectra from a small fraction of the gel spot. This technique shows promise for the direct identification of both parent and fragment masses of proteins contained in polyacrylamide gels.

Animals↗