摘要:

AbstractOptimal conditions of electroblotting that led to high protein recovery on polyvinylidene difluoride (PVDF) membranes were determined for sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). SDS concentrations in the gel and transfer buffer were found to be the most important factors affecting the amount of protein recovered on the PVDF membrane. The largest loss occurred during the first 10–30 min of transfer due to the relatively high initial SDS concentration in the gel. During this initial stage of transfer, most of the protein passed through the primary membrane and was partially retained on secondary and tertiary membranes. The value of presoaking gels prior to transfer to reduce the amount of SDS was evaluated by quantitating free SDS densitometrically and by correlating the reduced SDS concentration with increased electroblotting efficiency from presoaked gels. Transfer time was evaluated and no “overtransfer” was found even after very long transfer times. These results clearly indicate that proteins electroblotted onto PVDF membranes were tightly bound and could not be released by extending the transfer time. The effects of methanol and SDS concentrations on protein adsorption from solution to PVDF were also determined quantitatively. The results of this study strongly suggest that proteins fully saturated with SDS cannot bind efficiently to PVDF membranes. Since SDS is necessary for high protein mobility, the challenge in efficient electroblotting is to maintain an optimal SDS concentration which is high enough to permit effective removal from the gel and low enough to permit effective binding to the PVDF membrane. For 1.5 mm thick gels containing 0.2% SDS, presoaking the gel for 15–20 min in transfer buffer with 10% methanol prior to electroblotting provided the best recovery on the prima

ISSN:0173-0835    

DOI:10.1002/elps.1150130112

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

年代:1992

数据来源: WILEY

摘要:

AbstractMethods for the separation of peptides on a new type of thin‐layer chromatography (TLC) sheet and blotting onto polyvinylidene difluoride (PVDF) membranes with subsequent gas phase sequencing are described. For validation, the A and B chain of insulin were chromatographed on EmporeTMTLC sheets and either extracted or blotted onto PVDF membranes. The advantages and disadvantages of thin‐layer chromatography on Empore sheetsversushigh performance liquid chromatography (HPLC) are discussed, along with the possibility of combining the two methods. In addition, TLC was combined with electrophoresis (finger‐printing) for the separation of complex peptide mixtures. Blotting from TLC sheets onto PVDFmembranes was performed in two ways: contact diffusion and electrophoretic transfer. In our experiments electroblotting was more effective. Amino acid sequence determination of the B chain of insulin was possible both after extraction from the TLC sheet and after blotting onto PVDF membranes. In the former case, liquid phase sequencing and, in the latter case, gas phase sequencing was performed. The possibility to blot from TLC sheets onto membranes,e.g.PVDF, may prove useful in many fields, for example in biochemistry, and in molecular and cell bi

ISSN:0173-0835    

DOI:10.1002/elps.1150130113

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

年代:1992

数据来源: WILEY

摘要:

AbstractUsing polyacrylamide electrode gels during horizontal sodium lauryl sulfate‐polyacrylamide electrophoresis of radiolabeled proteins instead of electrode papers reaching into electrode buffer reservoirs, silver staining is improved and we reduce chemical and radioactive liquid waste during electrophoresi

ISSN:0173-0835    

DOI:10.1002/elps.1150130114

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

年代:1992

数据来源: WILEY

摘要:

AbstractA homogenizing system was developed to process multiple samples of various plant tissues. The equipment consists of a motor‐driven stainless steel homogenizer that fits perfectly into a hole of a disposable plate. Hard plant material like melon or squash seeds (maximal length of 1.4 cm) could be homogenized within 10 s by forcing the homogenizer towards the bottom of a 24‐ or 48‐well tissue culture plate. Smaller seeds could be homogenized within 10 s in a similar way using a 96‐well tissue culture plate. Leaf tissue, or other types of soft plant tissue, could be homogenized with the same equipment. For homogenization of small samples,e.g.pistils of small flowers, a 60‐well Terasaki plate was used. For homogenization of several hundreds of leaf tissue samples, an apparatus was developed that contains 12 homogenizers driven simultaneously by an electromotor. A tissue culture plate containing 96 leaf samples could be handled in this way in less than 10 min. The performance of the equipment was evaluated by analyzing homogenates of several types of plant tissue by different electrophoretic t

ISSN:0173-0835    

DOI:10.1002/elps.1150130115

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

年代:1992

数据来源: WILEY

摘要:

AbstractWhen plant tissue extracts are electrophoresed on polyacrylamide gels and the gels are stained for malate dehydrogenase by the standard NAD‐dependent dehydrogenase reaction, terminating in the formation of reduced Nitroblue Tetrazolium (NBT), achromatic bands, in addition to the expected chromatic bands, are observed. The achromatic bands are seen when the staining conditions favor a generalized background staining of the gel and have been shown, in a previous study, to be caused by peroxidase isozymes [1]. The present study examined the mechanism by which peroxidase produced the achromatic bands using horseradish peroxidase (HRP). The generalized background staining resulted from the phenazine methosulfate (PMS)‐mediated reduction of NBT. This reduction was enhanced by H2O2and suppressed by HRP. Peroxidase apparently catalyzes the peroxidative oxidation of reduced PMS, which suppresses the generalized reduction of NBT in gel regions containing peroxidase isozymes producing the achromatic bands. In contrast, however, HRP also appears to catalyze the peroxidative oxidation of reduced NAD, but this reaction increases the reduction of NBT. The results are discussed in the context of the mechanisms proposed by others for the PMS‐mediated reduction of NBT and for the peroxidase‐catalyzed NADH‐dependent formation of H2O2. This peroxidase‐catalyzed reaction has been proposed for the plant peroxidases involved in li

ISSN:0173-0835    

DOI:10.1002/elps.1150130116

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

年代:1992

数据来源: WILEY

摘要:

AbstractWe have demonstrated the feasibility of using bacteriophage ghost proteins, tritiated by metabolic labeling, as a set of standard markers for two‐dimensional gels in which the proteins are to be detected by silver staining. The results indicate that a 2.5 μg load of phage proteins yields a reproducible silver pattern of 48 spots. The spots can also be readily identified by radioautography and radiofluorography, establishing their value as a standard constellation of markers. Quantification of these patterns by computerized densitometry indicates a general agreement between detection by silver staining and detection by radiofluorograp

ISSN:0173-0835    

DOI:10.1002/elps.1150130117

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe total polypetide composition of mature grape berries was analyzed by one‐dimensional sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis and two‐dimensional electrophoresis (isoelectric focusing in the first dimension followed by sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis in the second dimension), followed by Coomassie Blue and nitrate silver staining, respectively. Adapted methods for total protein preparation of grapes and for two‐dimensional gel electrophoretic separation of polypeptides are presented. The grape patterns presented up to 52 fractions withMrs ranging from 15000 to 110000. The polypeptides displayed pIs from 4.6 to 7.3. A group of spots fromMr28000 to 83 000 and with a pIfrom 4.6 to 5.4 was strongly silver stained. TheMr28 000 spot, pI 4.6, was revealed to be a complex of four fractions. Reproducible separations were obtained with the different carrier ampholyte mixtur

ISSN:0173-0835    

DOI:10.1002/elps.1150130118

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

年代:1992

数据来源: WILEY

摘要:

AbstractA procedure for the analysis of glycoproteins and glycopeptides using the PhastSystem with detection by the periodic acid‐Schiff stain is described. Following sodium dodecyl sulfate or nondenaturing polyacrylamide gel electrophoresis and also isoelectric focusing, samples are stained directly for the presence of carbohydrates. By using the PhastSystem, the method is rapid, sensitive, reliable and allows storage of the gels without a change in the stain. As little as 0.1 μg of protein‐associated carbohydrates can be detected. The staining procedure is also used following isoelectric focusing of mucin‐derived glycopeptides to visualize their charge differences and the increase of their isoelectric point after neuraminidase tre

ISSN:0173-0835    

DOI:10.1002/elps.1150130119

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

年代:1992

数据来源: WILEY

摘要:

AbstractPolyacrylamide gel electrophoresis has become the most widely used separation method in biological science. Once electrophoresis is complete the protein bands must be localized prior to excision. A zig‐zag gel cutter is described which cuts a strip of gel from the side of a slab gradient gel or a gel of uniform concentration in peaks and valleys. The location of the protein of interest is determined by counting the number of peaks on the stained side strip. The portion of the unstained gel corresponding to the same count (number of valleys) is cut to recover the protein of interest from the main gel for further manipulation

ISSN:0173-0835    

DOI:10.1002/elps.1150130120

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

年代:1992

数据来源: WILEY

摘要:

AbstractWe describe a modification of transverse pore gradient gel electrophoresis in which pore size is regulated by crosslinker proportion (%C) rather than by total monomer concentration (%T). We electrophoresed plasma lipoprotein [a] transversely across linearN,N′‐methylenebisacrylamide gradients and measured mobility as a function of %C. This method allows for the simultaneous assessment of pore sizes generated over a wide range of crosslinker proporti

ISSN:0173-0835    

DOI:10.1002/elps.1150130121

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

年代:1992

数据来源: WILEY