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PubMed1996

Direct isolation of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analysis by electrospray-ionization mass spectrometry.

A new method for the isolation of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using electroelution with a modified buffer system, is described. The method is suitable for direct characterization by electrospray-ionization mass spectrometry (ESI-MS), or alternatively matrix-assisted laser desorption-ionization (MALDI), no additional purification steps being required. Following separation by SDS-PAGE, proteins were stained with Coomassie Blue, and isolated by electroelution using SDS-free ammonium acetate (pH 2.5) as elution buffer. The polarity of the electroelution system was reversed, which, upon in situ dissociation of protein-SDS complexes, resulted in migration of free proteins to the cathode under the conditions employed. Recovery rates of 25-58% were determined for model proteins. Analyses by ESI-MS provided exact molecular weight determinations of isolated proteins and of protein mixtures not resolved by SDS-PAGE. Essentially SDS-free molecular ions were obtained, except for bovine serum albumin with one SDS-adduct. Charge distribution of molecular ions were similar to those of the native proteins. The effects of beta-mercaptoethanol (beta-ME) and dithiothreitol (DTT) during electrophoresis were studied with hen egg white lysozyme, revealing the formation of mixed disulfide adducts between proteins and reducing agents. In a first bioanalytical application, hemofiltrate proteins from a patient with uremia were separated by SDS-PAGE. An ESI-MS analysis of the proteins isolated from the two most intensive gel bands enabled exact molecular weight determinations, and demonstrated that a gel band of ca. 17 kDa consisted of two different proteins.

Amino Acid Sequence↗
PubMed1996

Intrinsic curvature of plasmid DNAs analyzed by polyacrylamide gel electrophoresis.

The electrophoretic mobility of two small DNA plasmids, pUC19 and Litmus 28, linerarized by digestion with a variety of single-cut restriction enzymes, has been studied. The permuted sequence isomers migrate with identical mobilities in agarose gels, as expected, but exhibit different mobilities in large-pore polyacrylamide gels, suggesting that the parent plasmids contain sequence-specific sites of curvature and/or anisotropic flexibility. Both plasmids contain apparent bend centers near their origin(s) of replication; pUC19 also has a major apparent bend center near the promoter of the ampicillin resistance gene. These apparent bend centers are observed under a variety of experimental conditions, suggesting that they correspond to sites of stable curvature in the parent plasmids. Both plasmids also contain minor bend centers that are observed under a sub-set of electrophoretic conditions and disappear when divalent cations are added to the solution, suggesting that these apparent bend centers may correspond to localized regions of variable flexibility.

DNA, Circular↗
PubMed1996

Mechanisms of retardation of rigid spherical particles with 3 to 1,085 nm radius in capillary electrophoresis, using buffered polyacrylamide (molecular weight 5 x 10(6)) solutions.

Subjecting particles in the size range of 3 to 1085 nm radius (R) to capillary electrophoresis in buffered solution of entangled uncrosslinked polyacrylamide (M(r) 5 x 10(6)), it was found that particle size-dependent retardation ("molecular sieving") becomes electric field- and particle size range-dependent once the particle size exceeds 15-20 nm in radius. The field strength dependence of the retardation coefficient [KR = d(log mobility)/ d(polymer concentration] and the positive or negative sign of dKR/dR suggest the existence of two different mechanisms of molecular sieving depending on the particle size range: particles with diameters less than the screening length (or blob size) of the polymer network are thought to penetrate into the available spaces within a discontinuous polymer network; particles with diameters larger than the screening length (or blob size) of the polymer network are thought to undergo size-dependent retardation by exerting shear stress against polymer chains, and displacing them, so as to cause local deformations in a continuous polymer network. A limit in the separating capacity of molecular sieving, due to a sharp increase in the rate of band widening with polymer concentration, was found when the value of the retardation coefficient exceeded 60 (mL/g).

Acrylic Resins↗
PubMed1996

Sodium dodecyl sulfate-polyacrylamide gel electrophoretic method for assessing the quaternary state and comparative thermostability of avidin and streptavidin.

Avidin, a positively charged egg-white protein, aggregates extensively when mixed at ambient temperatures with anionic detergents, such as sodium dodecyl sulfate (SDS). The resultant aggregates fail to penetrate the stacking gel during polyacrylamide gel electrophoresis (PAGE). To prevent the formation of such aggregates, avidin was acetylated and the pI was thus reduced. Acetylated avidin was found to behave in a manner similar to that of streptavidin; under nondenaturing conditions (i.e., incubation of samples at room temperature), both proteins normally migrated mainly as tetramers with a tendency to form oligomers of the tetramer. When samples were boiled, both proteins migrated mainly as the monomer. The comparative stability properties of avidin and streptavidin were also examined using SDS-PAGE by heating samples and determining the extent of dissociation of tetramers to monomers as a function of temperature. A distinctive transition temperature could be defined for individual samples. Using this assay, it was determined that, in the absence of biotin, the quaternary structure of streptavidin is more stable than that of avidin. Biotin appears to stabilize structures of both avidin and streptavidin to a similar degree. Acetylation of avidin thus provides a simple means to analyze the quaternary structure of the molecule using SDS-PAGE.

Avidin↗
PubMed1996

A taxonomic study of the genus Listeria using sodium dodecyl sulphate-polyacrylamide gel electrophoresis of intracellular proteins.

Fifty-one strains of Listeria spp. and one strain of Jonesia denitrificans were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and the resulting histograms compared using the unweighted pair group method with averaging. The majority of L. monocytogenes strains formed a discrete cluster with a minimum correlation of 73% similarity. The position of strain NCTC 11994 within the main L. monocytogenes cluster supports the recommendation by the National Collection of Type Cultures that it, rather than NCTC 10357, be adopted as the type strain of the species. All L. innocua strains formed a discrete cluster with a minimum correlation of 85% similarity. The similarity shown by L. grayi and L. murrayi, both between each other and with the other Listeria strains tested, support their designation as a single species within the genus Listeria. Although the single strain of J. denitrificans showed the least similarity with the other strains it was not sufficient to warrant support for its removal from the genus Listeria. This limited study provides a different perspective both between species in the genus Listeria and between the genera Listeria and Jonesia.

Bacterial Proteins↗
PubMed1997

DNA mobility anomalies are determined primarily by polyacrylamide gel concentration, not gel pore size.

The dependence of DNA mobility anomalies on gel pore size has been studied in polyacrylamide gels with a wide variety of compositions, using molecular weight ladders containing multiple copies of normal (12B) and anomalously slowly migrating (12A) 147-base pair restriction fragments from plasmid pBR322 as the migrating probe molecules. If the gel pore size is increased by decreasing the total acrylamide concentration (%T) at constant cross-linker ratio (%C), the usual method of increasing gel pore size, the mobility anomalies decrease with increasing gel pore radius as though the 12A multimers were retarded by a sieving mechanism. However, the decrease in the mobility anomalies is independent of whether the apparent gel pore radius is larger or smaller than the DNA radius of gyration, suggesting that gel pore size is not the controlling variable. If the acrylamide concentration is held constant and the gel pore size is increased by decreasing %C at constant %T, the mobility anomalies of the largest 12A multimers (6 mers and higher) decrease with increasing gel pore radius, because of sieving effects, until the effective gel pore radius becomes approximately equal to the DNA radius of gyration, after which the mobility anomalies level off and become independent of gel pore size. The mobility anomalies exhibited by 5-mers and smaller multimers of fragment 12A are independent of gel pore radius in all gels with constant %T. Similar results are observed with a molecular weight ladder containing phased A-tracts from the kinetoplast bending locus. Since the anomalous electrophoretic mobilities depend primarily on the total acrylamide concentration in the gel, and not on the apparent gel pore radius, increases in the magnitude of the mobility anomalies with increasing gel concentration (and decreasing gel pore radius) cannot be taken as evidence for DNA curvature.

Acrylic Resins↗
PubMed1997

Resolution of high molecular weight proteins in dependence on electric field strength in polyacrylamide gel electrophoresis.

Resolution of high molecular weight proteins, in the upper region of polyacrylamide gels, was studied in relation to the type of electric field. Separations by constant field gel electrophoresis (CFGE) were compared to those in pulsed oscillatory high-performance electrophoresis (POPE), a novel technique which allows electrophoresis at high field strengths owing to a novel local field distribution. This distribution contributes to structural and mechanical stability of the gel with resultant well-reproducible separation, enhanced resolution, and higher absolute mobility of proteins in POPE.

Electricity↗
PubMedDate not supplied

Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: implications for proteome mapping.

Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.

Acrylic Resins↗
PubMed1997

Reorientation of large DNA molecules in concentrated polyacrylamide solution during crossed-field electrophoresis.

Recently, we found that, in concentrated neutral solutions, DNA molecules migrate in linear conformation under steady electric field. In this paper, we report the conformational change of DNA during 120 degree crossed-field electrophoresis in the same polymer solution. We found that, in concentrated polyacrylamide solutions, the reorientation process of DNAs becomes simple: the DNA goes back along the previous track and the reorientation time is longer for larger DNA. Such a backtrack motion has been thought to be an essential motion for the separation of DNA fragments in pulsed field gel electrophoresis. We expect that this phenomenon is useful for a more efficient separation technique of large DNAs than the current pulsed field gel electrophoresis.

Acrylic Resins↗
PubMed1997

The effect of sodium tetradecyl sulfate on mobility and antigen detectability of microtubule proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Several factors been reported to influence the mobility of polypeptide in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) including the brand of SDS. Using microtubule proteins from axonemes of Lytechinus pictus and Spisula solidissima sperm and meiotic spindles of Spisula solidissima we demonstrate that the change in mobility was caused by sodium tetradecyl sulfate (STS), a major contaminant of many commercial SDS brands. We also examined the use of sodium tetradecyl sulfate and different SDS brands as a tool in extracting more information from immunoblot studies. Commercial SDS containing contaminants other than sodium tetradecyl sulfate reduced or eliminated the immunosignal from certain polypeptides and the loss of antigenicity could not even be recovered by immunoblot under "renaturing" conditions. It can thus be concluded that STS can be useful in separating and identifying comigrating polypeptides and in detecting additional immunobands in immunoblots.

Animals↗
PubMed1997

Identification of bacteriophage T4 virion proteins by transverse pore-gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dual amino acid labeling.

We have developed a horizontal N,N'-methylenebisacrylamide (Bis) acrylamide gradient sodium dodecyl sulfate (SDS) gel system that permits the evaluation of the purity of individual protein bands in complex mixtures. A Bis gradient gel is poured vertically and, after polymerization, reoriented horizontally. A single large sample spanning the top of the gel is then run down at right angles to the gradient. The relative mobility of a few proteins varies considerably from the rest, causing them to merge with and cross other bands as the Bis concentration changes. Band splitting revealed that several bands previously thought to represent a single species are actually comprised of comigrating proteins. Once the Bis/monomer concentration offering the best separation was identified, we sought a simple method for identifying the genetic origin of bands, since many proteins now migrated in new positions on the gel. We reasoned that if infected cells were simultaneously labeled with [35S]methionine and [3H]leucine and the purified virion proteins analyzed to determine their 35S/3H ratio, we could identify most proteins by comparing this ratio with one calculated from the T4 DNA sequence. Our expectations were realized, and we here report the separation and identification of all T4 virion proteins. Finally, we comment on the incorporation of various changes to the original Laemmli SDS-polyacrylamide gel formulations that have been reported in the literature.

Bacteriophage T4↗
PubMed1998

Activity staining on polyacrylamide gels of trypsin inhibitors from leaves of sweet potato (Ipomoea batatas L. Lam) varieties.

The failure of activity staining of trypsin inhibitors in crude leaf extracts of sweet potato varieties including Tainong 27, Tainong 34, and Tainong 57 on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel was prevented by dipping the gels in solutions containing 10-40 mM hydrogen peroxide, 10 mM Tris buffer (pH 7.9) for 30 min before staining.

Acrylic Resins↗
PubMed1998

Elution of glycoproteins from replicas of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.

A method for the elution of glycoproteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) replicas of gels on polyvinylidene difluoride (PVDF) membranes is described. Ten model glycoproteins were resolved by SDS-PAGE and then electrotransferred onto PVDF membranes. After reversible staining, glycoprotein bands were eluted with a mixture of SDS/Triton X-100 at pH 9 or with a mixture of guanidinium hydrochloride/lysophosphatidylcholine at neutral pH. For both types of eluents, the final recoveries ranged from over 30% to about 80%. Good recoveries and mild conditions of elution render the method applicable for the structural elucidation of glycan chains.

Acrylic Resins↗
PubMed1998

The gel edge electric field gradients in denaturing polyacrylamide gel electrophoresis.

It has previously been shown that zones of higher electric field form close to the loading end of the gel during denaturing polyacrylamide gel electrophoresis. Here we show that the field can reach up to three times its normal mean value a few cm in front of the loading wells when 44.5 mM Tris-44.5 mM boric acid-1 mM EDTA is used as the gel buffer. We also demonstrate that this electric field gradient is mostly due to the difference in ion transference numbers at the gel/buffer interface caused by the high viscosity of the urea solution contained in the gel. This field gradient leads to increased band widths and forces us to redefine both the electrophoretic mobility and the mean field intensity. We discuss some methods that can be used to minimize the effects of this gradient.

Acrylic Resins↗
PubMed1998

Detection of clonal T cells in cutaneous T cell lymphoma by polymerase chain reaction: comparison of mutation detection enhancement-polyacrylamide gel electrophoresis, temperature gradient gel electrophoresis and fragment analysis of sequencing gels.

Cutaneous T cell lymphomas (CTCL) can be differentiated from benign inflammatory dermatoses by the demonstration of clonal T cells in skin biopsy. As a marker of the T cell clonality, the rearrangement of the T cell receptor (TCR) genes is amplified by polymerase chain reaction (PCR) and subsequently analyzed by several electrophoresis techniques. Since the validity of this approach depends substantially on the separating capacity of the applied electrophoresis technique, we investigated in the present study the lower detection limit and the sensitivity of heteroduplex-loaded polyacrylamide gel electrophoresis on MDE (mutation detection enhancement) gels (HD-MDE PAGE), of heteroduplex-loaded temperature gradient gel electrophoresis (HD-TGGE) and fragment analysis (FA) on sequencing gels. Genomic DNA from formalin-fixed, paraffin-embedded skin biopsies of 53 CTCL specimens and 27 samples of benign dermatoses was analyzed by TCRy PCR followed by electrophoretic separation. Clonality was detected by HD-MDE PAGE in 22, by HD-TGGE in 34, and by FA in 33 of the 53 CTCL cases. Additionally, FA revealed an oligoclonal fragment profile in seven CTCL specimens. In the 27 samples from benign dermatoses, HD-MDE PAGE and HD-TGGE showed the expected polyclonal pattern in 26, and FA in 25 specimens. HD-TGGE and FA detected a clonal pattern down to a dilution of 10(3) monoclonal cells in 10(6) peripheral blood mononuclear cells (PBMC), while HD-MDE PAGE revealed a detection limit of 10(4) monoclonal cells in 10(6) PBMC. In conclusion, HD-TGGE and FA possess a higher sensitivity and lower detection limit than HD-MDE PAGE. Therefore, both former techniques are useful tools for the routine diagnostic procedure. With regard to time and cost, we recommend HD-TGGE.

Cell Separation↗
PubMed1998

DNA cycle sequencing of a common restriction fragment of Staphylococcus aureus bacteriophages by capillary electrophoresis using replaceable linear polyacrylamide.

The nucleotide sequence of a part of a 4.9 kbp common restriction fragment isolated from Staphylococcus aureus bacteriophage (bacterial virus) 3A has been determined by capillary electrophoresis (CE). The fast separation of sequencing fragments in linear polyacrylamide solution at a temperature of 55 degrees C allowed the reading of more than 650 bases of sequence in 60 min. The single strand (ss)DNA fragments were prepared by cycle sequencing with fluorescently labeled dideoxy-terminators on the cloned bacteriophage DNA template. With respect to analysis speed, sequence read-length, low sample consumption and automation, CE offers a simple, labor-saving and inexpensive procedure for DNA sequencing. Operating the CE columns at elevated temperature proved to be a rapid procedure capable of extending sequence read-length. The resulting sequence of the common restriction fragment can be used for the preparation of specific primers and oligonucleotide hybridization probes for identification of Staphylococcus aureus bacteriophages and/or prophages belonging to the bacteriophage species 3A.

Acrylic Resins↗
PubMed1998

Use of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) for routine identification of enzymatically digested proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Automated liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis of >100 tryptic digests carried out on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separated, Coomassie Blue-stained proteins that were prepared by >50 different laboratories demonstrates that a commercial electrospray/quadrupole ion trap mass spectrometer and the tandem mass correlation algorithm developed by Eng et al. (Am. Soc. Mass Spectrom. 1994, 5, 976-989) provide an extremely robust and facile approach to routine protein identification. By requiring a minimum of two significant matches to peptides that would be predicted to be produced by the protease that was used, low pmol levels of proteins can be identified with high confidence while minimizing the probability of identifying the protease itself and/or the ubiquitous contaminant, keratin. Hence, in only 7% of the digests analyzed was keratin identified and in only 5% of the digests analyzed was the protease itself identified. In contrast, 58% of the analyzed samples were identified and, in many instances, multiple proteins were identified in the same sample. Although the median amount of digest analyzed was 6.1 pmol, the limit of sensitivity (as the instrument is configured with a flow rate of 4 microL/min) appears to be at the 500 fmol level. Since one of the primary reasons for not identifying a sample is that its sequence is not yet in the database searched, the utility of an LC MS/MS approach to protein identification will certainly increase in the future as the sequences of more genomes are completed.

Chromatography, High Pressure Liquid↗
PubMed1998

Migration of single-stranded DNA in polyacrylamide gels during electrophoresis.

By using fluorescence recovery after photobleaching (FRAP) and electric birefringence, the migration of single-stranded DNA in polyacrylamide gels and orientation as a response to an electric pulse were investigated. Electrophoretic mobility is in good agreement with the model of biased reptation including fluctuations. The determination of the electrophoretic mobility in solution, mu0, allows an estimation of the gel pore diameter seen by the molecule. As previously observed for double-stranded DNA, the electric birefringence results from two processes: the alignment of the molecule along the electric field and the elongation of the primitive path in the gel, for long single-stranded DNA (>2000 bases). The combination of results obtained with the two techniques allows us to propose experimental conditions to improve the separation of single-stranded DNA with pulsed field techniques.

Acrylic Resins↗