摘要:

AbstractThe problem of (thermal) band broadening during DNA gel electrophoresis is studied analytically and numerically using the reptation model. It is shown that the orientation of the end‐to‐end vector of the molecules leads to increased diffusion and even to molecular size‐independent band broadening. Thus, the Einstein relation between mobility and diffusion holds for only very small molecules. Also, “self‐trapped” molecules are predicted to give fuzzy bands, as observed experimentally. Finally, megabase molecules are discussed and the consequences of our predictions for the improvement of sequencing are

ISSN:0173-0835    

DOI:10.1002/elps.1150140102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractA theory for the gel electrophoresis of a flexible polyelectrolyte, bearing an uncharged bulky label or an uncharged section at one end, is presented. We first consider a gel that is fully permeable to the label: we calculate the degree of stretching of the polyelectrolyte and its mobility as a function of chain size, electric field and label friction. Various regimes are identified, and their “existence domains” are calculated. For increasing friction, we predict a transition from a mobility decreasing with chain size to a mobility increasing with chain size. Secondly, we consider the possibility that the label may get trapped at some locations of the gel, a situation relevant to a method of “trapping electrophoresis” recently proposed by Ulanovskyet al. for DNA sequencing. A molecular model for detrapping by thermally activated “backward reptation” is constructed and solved using the Kramers rateequation theory. Different closed analytical expressions and approximate scaling laws corresponding to different regimes of stretching and field strengths are predicted. The most striking result is a mobility which exponentially decreases past a critical size N p*, which decreases with increasing field. In the regime relevant to the experiments by Ulanowskyet al., we predict N p*∼ E−2/3. The predictions are in good qualitative agreement with presently available experiments, but further experimental investiga

ISSN:0173-0835    

DOI:10.1002/elps.1150140103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe efficiency of size separations of polystyrene sulfate, 120‐1085 nm radius, by molecular sieving in polymer solutions, expressed by the separation efficiency functionS=MOdKR(R)/dRTe−KR(R)T[whereMOis the mobility in free solution,KRthe retardation coefficient,Rthe geometric mean radius of the particle,KR(R) the retardation coefficient as a function ofR, and the polymer concentration isT] increases from methylhydroxypropyl cellulose to polyvinyl alcohol to uncrosslinked polyacrylamide to agarose above its gelling temperature. Separations of DNA, in the size range of 3–21 nm radius, are by at least one order of magnitude more efficient than those of polystyrene sulfate in the size range of 120–1085 nm radius. A plot ofS vs. Rin the experimental range ofTis constructed for the four polymer solutions; this allows one to select optimal media and concentrations for the sieving of particles in the desired range of molecula

ISSN:0173-0835    

DOI:10.1002/elps.1150140104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractIn vitroamplification of the alphoid repeated sequences clustered in the centromeric regions of the human × and Y chromosomes was carried out. A modification of the amplification conditions reported by Witt and Erickson [1] gave clear amplification bands from 10 pg of genomic DNA template. Results were obtained from the following aged tissue samples: 1 μL three‐year‐old blood stain, one‐year‐old single hair roots, six‐month‐old saliva stains and twelve‐year‐old bone fragments. Good results were obtained even when the template was artificially fragmented into fragments o

ISSN:0173-0835    

DOI:10.1002/elps.1150140105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractDNA digest analysis with polymer‐filled capillaries in capillary electrophoresis is described. The samples analyzed consisted of commercially available standards including a 100 base pair ladder with repeating units up to 2000 base pairs. Three DNA digests covered the most common small fragment ranges: up to 600 base pairs, up to 1500 base pairs and from 100 to 2500 base pairs. All samples were separated by traditional slab gel electrophoresis at various agarose concentrations and by automated capillary electrophoresis. The capillary electrophoretic separations were achieved with noncross‐linked polyacrylamide from 6% monomer solutions. The acrylamide was polymerized inside the capillary, which was coated with a methacryloxysilane to insure binding of the polyacrylamide to the capillary wall. With 6% columns, excellent separations were observed up to 600 base pairs with base line resolution for fragments differing in less than 10 base pairs. Resolution power for fragments between 600 and 2200 decreased to about 300 base pairs. Compared to slab gels, capillary electrophoresis achieved better resolution in the low fragment range, whereas with the reported column composition, slab gels were superior above 600 base pairs. Fast access to the analysis of a specific sample, automation, and a larger dynamic range for sample load are further benefits of a DNA digest analysis by capillary electrophore

ISSN:0173-0835    

DOI:10.1002/elps.1150140106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractA new method of capillary electrophoresis was established to separate the three cresol isomers. Cresols are of importance due to their use as insecticides, precursors of various dye intermediates, resins, fire‐resistant fluids and other purposes. Reversion of the electroosmotic flow was carried out by a polycationic ammonium compound. The separations were carried out at high pH values (pH 12) to obtain complete dissociation of the cresols and short times of analysis (less than 5 min) through fast migration of the cresol towards the anode. Various aliphatic alcohols were added to improve peak shape and resolutio

ISSN:0173-0835    

DOI:10.1002/elps.1150140107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractPhotopolymerization of polyacrylamide gels in the presence of methylene blue (100 μM) and a redox couple (1 mMsodium toluenesulfinate, a reducer, and 50 μMdipheriyliodonium chloride, an oxidizer) has been investigated. The gel point,i.e.the time needed for onset of gelation upon illumination, has been found to lengthen progressively at lower temperatures and at lower light intensities. If the three catalysts are progressively diluted, the gel point does not vary for a threefold dilution, but gelation is greatly hampered below a 1:5 dilution of the three effectors. Photobleaching has been assessed as a function of liquid layer thickness (from 0.5 to 2 mm), of a progressive dye dilution (down to a fourfold dilution) and as a function of temperature. A maximum of elastic modulus is located in correspondence to a minimum of permeability (both situated at 5% cross‐linker). It is found that methylene blue‐activated polymerization produces polyacrylamide gels with elastic properties which are higher than in persulfate‐activated gels, so far the most popular matrices for electrokinetic separations. Due to the ease of preparation, the full control of all experimental parameters, and the lack of oxidizing power of this catalyst system (as opposed to the strong oxidation power of persulfate catalysis), methylene blue catalysis is advocated as a valid alternative to other redox

ISSN:0173-0835    

DOI:10.1002/elps.1150140108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractIn most silver staining methods the first step in the staining of proteins separated after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis is a rather protracted fixation of the gels. Optimum fixation should be short, cause no background staining and effectively immobilize the proteins in the gel without masking the proteins for reaction with the staining solution. Further, the concentration of the fixing compounds should be as low as possible due to the potentional toxicity of fixatives. Fixation for only 5 min with mixtures of very low concentrations of formaldehyde and glutaraldehyde in ethanol, or a solution of formaldehyde or glutaraldehyde in picric acid and ethanol, fulfill these demands, provided that the gels were prefixed in ethanol‐acetic acid for 10 min. As a consequence of these results a fast and sensitive silver staining procedure is propo

ISSN:0173-0835    

DOI:10.1002/elps.1150140109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractGiant proteins in the megadalton range (>0.5 MDa) appear to play important structural and functional roles in striated muscle. Titin (∼ 3 MDa) is involved in the generation of resting tension and the assembly and stability of the sarcomere in skeletal and cardiac muscle tissues, while nebulin (∼ 0.7 MDa) is thought to regulate thin filament length in skeletal muscle. Sodium dodecyl sulfate (SDS)‐gel electrophoresis is an important tool in revealing the size, quantity and integrity of these giant proteins in muscle tissues. We report here a method for solubilizing, detecting and quantifying titin and nebulin from short segments of single fibers of the rabbit psoas muscle. Muscle proteins ranging from 15 kDa to 3MDa were resolved on 3.3–12% gradient polyacrylamide gels that were silver‐stained and quantitated by densitometry. Presoaking fiber segments in a low ionic strength pH 8.4 buffer enhances the amount of solubilized titin and nebulin. Solubilizing the presoaked fiber segments with SDS at 60°C for 60 s maximizes the amount of intact titin; solubilizing at higher temperatures causes extensive degradation of titin. Detection sensitivity is sufficient to study titin and nebulin in fiber segments as short

ISSN:0173-0835    

DOI:10.1002/elps.1150140110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe use of Nile Red to track and stain low density lipoprotein (LDL) and modified LDL in agarose gels was investigated. Lipoproteins were prestained with Nile Red, a fluorescent dye, prior to electrophoresis. After 2 h of electrophoresis, the LDL and modified LDL were visualized using a UV transilluminator with an excitation wavelength of 302 nm. Spectrofluorometric analysis revealed that the Nile Red fluorescence of the stained LDL had an emission maximum of 609 nm. This rapid staining method of LDL and modified LDL can detect as little as 2.5 μg of LDL protein and permits the immediate visualization of these lipoproteins in agarose gels

ISSN:0173-0835    

DOI:10.1002/elps.1150140111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY