摘要:

AbstractProtein profiles of skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) and normal individuals have been compared using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS) combined with various detection systems. Protein patterns of normal and DMD fibroblasts obtained usin 10 % polyacrylamide gels showed no qualitative or quantitative differences. The use of acrylamide gels results in increased resolution. No qualitative or quantitative differences were observed using Coomassie Brilliant Blue R‐250 staining. However, Coomassie Brilliant Blue lacks the sensitivity to detect many minor components. Therefore a silver staining procedure was used, which resulted in greatly increased detection sensitivity. Background staining did not increase with gel concentration, but stain development proceeded more slowly at higher acrylamide concentrations. No qualitative differences were observed between normal and DMD profiles. Fibroblasts radiolabelled with [35S]‐methionine were analysed using gradient gels. No qualitative differences between DMD and normal patterns were observed by autoradiography of dried gels. However, quantitative analysis of slicing of gel rods revealed differences between normal and DMD fibroblasts. Two methods of statistics were used to analyse this data, firstly a method that accounts for inter‐experimental variation (termed “batch statistics”) and secondly the standard t‐test. Regions of 73 000 to 68 000 and 48 000 molecular weight were decreased in DMD samples and this finding was significant by both methods of statistical analysis. Regions of 175 000 and 53 000 molecular weight were significantly elevated in DMD preparations only by t‐test, whereas a regions of 32 × 103molecular weight was only significantly elevated when tested by ba

ISSN:0173-0835    

DOI:10.1002/elps.1150030402

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

年代:1982

数据来源: WILEY

摘要:

AbstractProtein profiles of skin fibroblasts labelled with [35S]‐methionine from patients with Duchenne muscular dystrophy (DMD) have been compared with those of cells from normal individuals by isoelectric focusing on thin polyacrylamide gels containing denaturing and solubilising agents cast on silanized supports. The routine method of sample solubilisation used a mixture of 8 M urea and 2 % NP‐40. The inclusion of sodium dodecyl sulphate (SDS) in the sample was found to result in removal of non‐ionic detergent from the gel even if a competition step had been used. In the presence of SDS less material remained at the point of sample application with a concomitant increase in bands in the middle region of the gels. Gels containing a mixture of sulfobetaine and NP‐40 were found to be inferior to those with 8 M urea and 2 % NP‐40, and high levels of urea were found to precipitate the zwitterionic detergent. No consistent qualitative differences between normal and DMD patterns were observed using any of the solubilisation methods on pH 3 to 10 or pH 8 to 10.5 gels. In order to facilitate quantitative analysis by gel slicing techniques, a plastic sheet was developed which would reliably bind acrylamide in the presence of 8 M urea and 2 % NP‐40. The quantitative analysis revealed significant differences between normal and DMD profiles, but the relevance of these changes to the disease state awaits further in

ISSN:0173-0835    

DOI:10.1002/elps.1150030403

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

年代:1982

数据来源: WILEY

摘要:

AbstractDeposition of silver or silver compounds in proteins separated in linear gradient polyacrylamide electrophoretic gels is influenced by protein concentration and location, polyacrylamide concentration, extent of washing after incubation in ammoniacal silver hydroxide solution, and development time in a citric acid: formaldehyde solution. Our modification of existing silver staining methods provided routine resolution of polypeptides separated in 1.0 to 1.5‐mm‐thick gels without high background staining or surface staining and with picogram detection sensitivity. Removing electrophoretic components, such as sodium dodecyl sulfate (SDS), sulfhydryl reagents (2‐mercaptoethanol, dithiothreitol and dithioerythreitol), glycerol, tris‐glycine, carrier ampholytes, urea, and acetic acid, was essential in order to attain silver staining of proteins with a negligible background. Ammoniacal silver ions or silver ions, associated and unassociated with proteins separated in polyacrylamide gels, were removed during the washing step following incubation in ammoniacal silver hydroxide solution. Loss of stain intensity at discrete protein clusters was shown to correlate with polyacrylamide concentration in gradient gels. Standardizing the washing step enhanced reproducibility of staining. Using this silver staining method and computer‐assisted image processing, it was possible to detect quantitatively as little as 27 picograms of protein per mm in 10 %‐20 % linear gradient polyacrylamide gels (1.2 mm thick). Based on the lowest detection limits for Coomassie Brilliant Blue R‐250 staining of protein (10 nanograms), this method for silver staining is at least 370‐fold

ISSN:0173-0835    

DOI:10.1002/elps.1150030404

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

年代:1982

数据来源: WILEY

摘要:

AbstractA silver stain technique has been developed to study proteins in unconcentrated cerebrospinal fluid (CSF) after isoelectric focusing. This method is highly sensitive, and bands containing 25 to 50 ng protein can be clearly distinguished, so that small volumes (40 μl maximum) of native CSF can be used. Individual proteins (e. g., immunoglobulin G) can be detected easily by specific immunofixation. It is also possible to perform direct precipitation and direct specific immunofixation on a single gel in order to compare side by side the patterns of the whole and of specific proteins from different samples of CSF. The technique is simple and highly reproducible, and results are obtained 6 h after sample deposition (if immunofixation is used, a further 24 h washing is necessary). The sensitivity and versatility of this technique (immunofixation can be applied to the detection of any antigen) should permit its extension to other biological fluids with a low protein content

ISSN:0173-0835    

DOI:10.1002/elps.1150030405

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

年代:1982

数据来源: WILEY

摘要:

AbstractVertical slab gel electrophoresis of DNA with CH3HgOH‐containing agarose produces sharp bands whose mobilities are suitable for size estimation of single‐stranded DNA containing 600–20 000 bases. The relationship of electrophoretic mobility to size of DNA over this range is a smooth, S‐shaped function, and an empirical model was developed to express the relationship. The model involves terms in squared and reciprocal mobilities, and produced excellent fit of known standard markers to measured mobilities. It was used to estimate the sizes of six parvovirus DNAs: Kilham rat virus (KRV), H‐1, LuIII, and minute virus of mice (MVM) DNAs had molecular weights of 1.66 – 1.70 × 106, while the molecular weight of bovine parvovirus (BPV) DNA was 1.84 × 106and that of adenoassociated virus (AAV) DNA w

ISSN:0173-0835    

DOI:10.1002/elps.1150030406

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

年代:1982

数据来源: WILEY

摘要:

AbstractWhen [3H]‐labeled proteins are analyzed by polyacrylamide gel electrophoresis, the sensitivity of fluorograhic detection following gel impregnation with PPO (2,5‐diphenyloxazole) in DMSO (dimethylsulfoxide) is significantly reduced if the gel has been stained with Coomassie Brilliant Blue R‐250. Since the reduction in fluorographic efficiency depends on the concentration of stain, which in turn depends upon protein concentration, substantial errors may enter into quantitative measurements of fluorograms depending on sample composition. Another reduction in fluorographic efficiency results from reduced penetrability of the gels to PPO when a build‐up of methanol in DMSO and DMSO/PPO solutions is allowed. This loss of fluorographic sensitivity should be sample independent,

ISSN:0173-0835    

DOI:10.1002/elps.1150030407

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

年代:1982

数据来源: WILEY

摘要:

AbstractDiscontinuous isoelectric focusing in layers of granulated gels is used to separate up to 1000 mg protein with a resolution of 0.01‐0.015 pI. High resolution is achieved with (i) gel layers of reduced thickness (1 mm), (ii) high field strength in the final stage of focusing (100‐200 V/cm), (iii) long separation distances, and (iv) high Volthour (Vh) products ensuring steady state conditions. We describe approaches to rapid focusing based on either short separation distances or extensive prefocusing with resultant short residence times of the separated substances in the gel. The optimum load capacity is 3‐5 mg protein/ml gel‐bed volume while 10 mg protein/ml gle‐bed volume are still well tolerated. At high loading, major components are seen in the gel as transulcent zones which can be easily identified and harvested. Best results are obtained with Sephadex G‐200 (Superfine) or Bio‐Gel P‐60 (minus 400 mesh). Protein visualization with a new printing technique empolying cellulose acetate membranes requires only 2‐3 min and is insensitive to protein overloading and adhering gel particles. A simple and efficient method is described for the electrophoretic removal of carrier ampholytes from the focused proteins. High resolution preparative isoelectric focusing should prove attractive for many applications due to its great flexibility with respect to sample size, gel volume, separation time, protein recovery, ease of operation and

ISSN:0173-0835    

DOI:10.1002/elps.1150030408

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

年代:1982

数据来源: WILEY

摘要:

Abstractα1‐Antitrypsin was isolated from rat serum by salting out with ammonium sulfate in three steps, followed by repeated ion exchange on DEAE‐Sephadex A 50 and affinity chromatography on Affi‐Gel Blue. The fractionation was paced with isoelectric focusing in polyacrylamide gels with pH range 3.5‐9.5. The resultant protein was homogeneous in crossed immunoelectrophoresis using rabbit antiserum against whole rat serum. Isoelectric focusing in α1‐antitrypsin phenotyping plates with pH range 4–5 showed a clear five‐band pattern of isolated protein (isoelectric points 4.41, 4.48, 4.50, 4.58 and 4.61, respectively). Immunofixation and zymogram techniques were used to identify the α1‐antitrypsin bands. In sodium dodecyl sulfate/polyacrylamide gel electrophoresis two bands appeared. They had relative mobilities corresponding to apparent molecular weights of

ISSN:0173-0835    

DOI:10.1002/elps.1150030409

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

年代:1982

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150030410

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

年代:1982

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150030411

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

年代:1982

数据来源: WILEY