ISSN:0173-0835    

DOI:10.1002/elps.1150020302

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

年代:1981

数据来源: WILEY

摘要:

AbstractA color development system for staining polypeptides in one‐ and two‐dimensional polyacrylamide gel electrophoresis is described. The basis of the Process involves the complexing of silver with polypeptides reactive centers. The reaction is initiated by placing a polypeptide‐containing gel, previously equilibrated with an appropriate concentration of silver nitrate, into a reducing solution that contains sodium hydroxide, sodium borohydride, and formaldehyde. After an appropriate time in the reducing solution, the gel is equilibrated through two changes of an enhancing solution that contains sodium carbonate. The sodium carbonate is necessary for optimal color appear in the polypeptide ‐silver complexes after several hours in the enhancing solution and are best appreciated while viewing over a fluorescent light box that radiates light at 5000°K. The color of each polypeptide‐silver complex is clearly visible above the light background of the stained polyacrylamide gel. Colors of stained polypeptide are blue, green, yellow, and red. Subtle shades of colors also appear and thereby allow easy discrimination of overlapping spots of polypeptides in a two‐dimensional gel. To illustrate the method's relative sensitively, a two‐dimensional pattern of human fibroblast polypeptides is compared with patterns of a duplicate gel that is stained with Coomassie Blue and developed by autoradiography. The sensitivity of the silver stain process is superior to Coomassie Blue and is comparable to autoradiography after in corporation of conventional levels of35S‐ methionine. The utility of the procedure for identifying and characterizing human proteins is illustrated by staining human proteins is illustrated by staining human plasma and platelet polypeptides after two‐dimensional gel electrophoresis. The gel electrophoresis color development system consists of steps that are simple, reproducible, and sensitive, and most importantly, which yield colored polypeptide‐silver complexes that are reproducible from gel to gel

ISSN:0173-0835    

DOI:10.1002/elps.1150020303

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

年代:1981

数据来源: WILEY

摘要:

AbstractOptimal silver staining of proteins in polyacrylamide gels is a fine balance between silver deposition on the proteins and the level of background staining. Reproduciable staining requires empirical standardization of many steps in the procedure. Silver staining in the present form does not stoichiometrically stain proteins, unlike Coomassie Blue. Staining intensity does not bear a linear relationship to protein mass. This is demonstrated by densitometric evaluation of both silver and Coomassie‐stained marker proteins fractioned by sodium dodecly1 sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Although silver staining is more sensitive than Coomassie Blue, the increment in sensitivity is both different for individual proteins and dependent on protein concentration. Nevertheless it is an excellent method for one‐ and two‐dimensional (2‐D) electrophoresis if only minimal amounts of protein are available and only a qualitative or semi‐quantitative evaluation

ISSN:0173-0835    

DOI:10.1002/elps.1150020304

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

年代:1981

数据来源: WILEY

摘要:

AbstractA double one‐dimensional (D 1‐D) electrophoretic method for slab gels with polyacrylamide gel electrophoresis (PAGE) followed by polyacrylamide gel isoelectric focusing (PAGIF) is described for the demonstration of prealbumin (PA) from human sera. Almost pure PA is obtained when serum is submitted to Page i an anionic discontinuous (glycine‐chloride) buffer system at alkaline pH. After page the Bromophenol Blue‐stained tracking dye boundary in the gel containing PA is cut out and the gel strips are overlayed on an isoelectric focusing gel. A final PAGIF pattern of PA is obtained which is hidden by other serum proteins when one‐dimensional PAGIF is performed. PAGIF of PA in a gel containing a gradient from 0 to 8 M urea perpendicular to the pH axis reveals the conversion of at least 7 diffuse bands with isoelectric point (pI) values between 4.2 and 4.7 into three sharp zones with pI values of 5.45 for one minor zone and 5.7 for the two other zones lying very close together. Among 1900 sera from adult pregnant women, 2 were found containing a genetically determined PA variant with a pI of 5.9 for a minor and a pI of 6.35 for a more intense zone in addition to the usual pattern. From the variant PAGE and PAGIF patterns and their distribution in the families of the carriers, it is concluded that PA is a tetramer under control of an autosomal structural gene which remains stable during PAGE abut dissociates into the monomers during PAGIF in the presence or presence of urea it is followed that PA under physiological conditions possibly forms complexes with more components than expected from its known binding capacity for thyroxine and retinal binding protein (RBP). This highly resolving K 1‐D electrophoretic technique allows the simultaneous analysis of up to 96 samples under identical experimental conditions. It is concluded that PA may be a good candidate for human mutation monitoring at the pr

ISSN:0173-0835    

DOI:10.1002/elps.1150020305

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

年代:1981

数据来源: WILEY

摘要:

AbstractA method of testing batches of ampholytes is presented. By using carbamylated charges standards to co‐electrophorese with the protein sample in the first‐dimension isoelectric focusing gel, one can monitor, after running and staining the second‐dimension sodium dodecyl sulfate (SDS) slabs gel, the continuity of the pH gradient. Charge standards can also be used to check the reproducibility of the pH gradient among batches of ampholytes and to modify the new batch with a small amount of a narrow range ampholyte to assure reproducibility of experiments. Ampholytes for comparison were obtained from three major manufact

ISSN:0173-0835    

DOI:10.1002/elps.1150020306

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

年代:1981

数据来源: WILEY

摘要:

AbstractA simple and versatile optical technique for viewing discontinuities in the thickness of horizontal isoelectric focusing gels is described. The discontinuities in the gel are reproducible and relate directly to the ampholyte distribution. Characterization of the capture of the discontinuities in the gel is presented as well as a demonstration of how they may used for localization of focused proteins.

ISSN:0173-0835    

DOI:10.1002/elps.1150020307

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

年代:1981

数据来源: WILEY

摘要:

AbstractAlternative technical solutions of the concentration of protein samples or the recovery of proteins from gel slabs by displacement electrophoresis are demonstrated. The samples are collected at a boundary between high‐mobility leading ion and a low‐mobility terminating ion either in a small Sephadex G‐25 columns or in free solution in a rotating‐tube free electrophoresis apparatus. The boundary is kept stationary during electrophoresis by a counter flow of leading solution. Spacers and colored markers can be used to facilitate the collection of the proteins. Investigations showed that electroomosis is a very local phenomenon and that, as expected, the magnitude of the effects depends on the conductivity (field strength) of the solution. This implies that electroomosis will vary among different zones in a discontinuous system (as in displacement electrophoresis). Therefore, in most experiments om free solution it is very advantageous to arrange the system such that electrophoresis does no

ISSN:0173-0835    

DOI:10.1002/elps.1150020308

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

年代:1981

数据来源: WILEY

摘要:

AbstractUltrathin‐layer isoelectric focusing in polyacrylamide gels on silanized supports provided the means for developing a miniature system combining short focusing time with high resolution and operational simplicity. The essence of miniature ultrathin‐layer isoelectric focusing is the combined use of short separation distances (1–3 cm) and high field strengths (400–700 V/ cm), which can be applied in standard equipment simply by reducing the thickness of the gel to 20–50 um for improved heat dissipation. By the criterion of coalescence of proteins migrating from different positions, a steady state is reached in pH 4–9 servalyt T carrier ampholytes on 1 cm gels after 2 min (15 Vh) and on 3 cm gels after 10 min total focusing time (110–130 Vh). On 3 cm gels the resolution is 0.03–0.035 pI in wide‐range carrier ampholytes, and 0.01–0.02 pI in narrow‐range (0.3 pH units) carrier ampholytes isolated by preparative isoelectric focusing of pH 4–9 Servalyt T in Bio‐Gel P‐60 layers. Miniature ultrathin‐layer isoelectric focusing can easily be adapted to the analysis of multiple samples. When the volume is limited to 0.2–0.4 μl, up to four samples per cm can be applied directly on the gel surface. At this loading, the gel volume per focused sample amounts to only 0.5–5 μl. The gel volume per sample is reduced by a factor of 10–200, relative to the 10–12 cm separation distance in 50–100 μm gel layers. This results in a considerable saving of carrier ampholytes and reagents for gel preparation. For serial dilutions of pH marker proteins, the sensitivity of protein detection after staining with Serva Blue G or Serva Violet 49 is 10–15 ng protein in the miniature system. Miniature ultrathin‐layer isoelectric focusing should prove to be an attractive method for routine applications in clinical, industrial and research laboratories in those cases when only small amounts of material are available or when rapid results are required. The method is particularly suitable for studies of

ISSN:0173-0835    

DOI:10.1002/elps.1150020309

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

年代:1981

数据来源: WILEY

摘要:

AbstractWe have studied the use of bacteriophage T4 coat proteins as a complete set of molecular weight and isoelectric point markers for two‐dimensional electrophoresis. We present data which support the feasibility of this approach. The potential utility and advantages of this concept are discusse

ISSN:0173-0835    

DOI:10.1002/elps.1150020310

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

年代:1981

数据来源: WILEY

摘要:

AbstractIn a study of the biochemical mechanisms of renal toxicity of acetaminophen, quantitative studies of the nonspecific kidney esterases were carried out in New Zealand white female rabbits. After the administration of a sublethal dose of 2.5 g/kg acetaminophen, four animals each plus four controls were sacrificed at 24 and 48 h. subsequent isoelectric focusing of kidney homogenate supernatants over a pH range of 3.5–8.0 showed marked changes in each of the groups. Zymograms of the nonspecific esterases in each group showed a characteristic pattern which deviated greatly from control animals. A morphological and histochemical examination of kidney sections from all animals showed no pathological changes in any of the kidneys. This would indicate that abnormal biochemical changes are being manifested in the kidney long before any morphological deviations. It would seem that this group of enzymes could provide a valuable market for early toxicological change

ISSN:0173-0835    

DOI:10.1002/elps.1150020311

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

年代:1981

数据来源: WILEY