摘要:

AbstractImproved technologies or the synergistic use of complementary methods enhance the efficiency of research and permit the exploration of new approaches for the investigation of complex problems. High sensitivity protein sequence analysis and polyacrylamide gel electrophoresis are such complementary methods. Here we summarize the current status of high sensitivity sequence analysis of proteins separated in polyacrylamide gels and discuss strategies by which this technology can cnhance biological research by generating new approaches for the solution of complex, multifacetted problems. Finally, we outline lmminent technological advances in the area of high sensitivity protein sequence analysis and argue that further technological developments will ultimately lead to the generation of an integrated protein database (containing structural and functional as well as physiological information in an easily accessible form) of all the proteins separated by high resolution two‐dimensional gel electrophoresi

ISSN:0173-0835    

DOI:10.1002/elps.1150110702

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

年代:1990

数据来源: WILEY

摘要:

AbstractWe have used two‐dimensional gel electrophoresis as a general „preparative”︁ method to purify proteins for microsequencing analysis. In the first experiments, proteins derived from a total extract ofNicotiana tabacumleaf tissue were directly blotted from the gel onto poly(4‐vinyl‐N‐methylpyridinium iodide)‐coated glass fiber sheets. The major spots were excised and subjected to NH2‐terminal sequence analysis, which made it possible to identify five of the eight selected proteins, while two more were recognized by generated internal sequences. In a second set of experiments, proteins of human origin were separated on multiple two‐dimensional gels and the Coomassie Brilliant Blue‐stained spots were excised from the gels. The combined spots were re‐eluted and concentrated in a new gel and blotted on Immobilon. They were fragmented byin situproteolysis and the generated peptides were separated by reverse phase–high performance liquid chromatography and sequenced. At the average, the internal sequences that were obtained covered 35 residues per protein and allowed unambiguous identification of 13 of the 23 proteins analyzed so far. The sequence information obtained of the unidentified proteins is sufficient for further cloning. These results demonstrate that systematic sequence analysis of the major proteins seen in two‐dimensional gels is within the reach of current technologies. This offers a unique opportunity to link information contained in protein databases with known or fo

ISSN:0173-0835    

DOI:10.1002/elps.1150110703

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe fields of protein chemistry and molecular biology are currently merging for study of biologically relevant events and conditions. To obtain partial sequences of microamounts of protein, effcient integration of high resolution separation and sequencing technologies is required. We report here on improved methods that allow extensive internal sequencing of 10 to 20 picomoles protein recovered from one‐ or two‐dimensional gels. Each step of the standard protocol of Aebersoldet al.(Proc. Natl. Acad. Sci. USA1987,84, 6970–6974) and the required instrumentation were examined and specifically adapted for use with submicrogram amounts of protein. Optimizations ofin situmicrodigests and liquid chromatography were needed for improved peptide recovery. Subsequent automated sequencing required subpicomole analysis. New methods for S‐alkylation of gel‐separated proteins and accurate identification of tryptophan‐containing peptides were introduced to insure over all higher efficiencies. The acquired internal sequences facilitated cloning of the genes and several strategies are discussed. Applying our method, several proteins of unknown structure were sequenced and successfully identified or cloned. Internal sequences of submicrogram protein amounts, recovered from a single two‐dimensional gel ofEscherichia colitotal protein (120 μg), allowed unambiguous identification of the spots but pre‐gel enrichment will be required for analysis of most (90–95 %) other spots. Integration of comprehensive two‐dimensional gel protein databases with methods and strategies outlined here could potentially be an abundant source of DNA probes and markers useful for guidance of the human genome sequencing project and for analysis of the emerging

ISSN:0173-0835    

DOI:10.1002/elps.1150110704

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

年代:1990

数据来源: WILEY

摘要:

AbstractHigh‐resolution two‐dimensional polyacrylamide electrophoresis (2‐DE) is commonly used as an analytical approach to resolve and detect most of the numerous protein species of an organism. However, the isolation of microgram amounts of protein in a 2‐DE spot in a form suitable for microsequence analysis and amino acid composition analysis is a key step in the chemical characterization of these proteins. With the development of chemically inert membranes it is now possible to retain proteins present in low quantities from the polyacrylamide matrix with high yields. The immobilized proteins are suitable for direct sequence analysis and amino acid composition analysis. The combination of protein chemical and electrophoretic techniques makes it possible to obtain chemical information from subpicomole quantities of protein, resulting in the availability of a new set of biologically important proteins for structural analysis. This paper summarizes the methods and strategies for the chemical protein analysis of 2‐DE spots in our l

ISSN:0173-0835    

DOI:10.1002/elps.1150110705

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe use of new membranes such as activated or derivatized glass fibers as well as synthetic membranes, which are compatible with the hazardous sequencing reagents, are described. Precautions to be taken in order to preventN‐terminal block age of the proteins during electrophoresis and blotting are described, as well as the conditions for protein detection after blotting and protein treatment forin situamino acid analysis, fragmentation and microsequencing. For a number of standard proteins and bacterial ribosomal proteins microsequence analysis is reported for two commercially available sequencers (Applied Biosystems and Knauer

ISSN:0173-0835    

DOI:10.1002/elps.1150110706

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe technique of electroblotting polypeptides onto Polybrene‐treated glass fiber filter discs after protein detection with potassium chloride is evaluated further with different proteins in separate applications. The number of proteins analyzed with this method is now more than double that previously reported. Reproducible results in good yield are obtained. Average overall yield – including the electrophoretic step before blotting – is 26 %, with maximal recoveries through all steps up to 60 %. High sensitivity radiosequence analysis is also applicable. Recent modification of the previously described procedure include use of Whatman glass fiber filters, removal of air in the Polybrene‐impregnated filters by buffer penetration under reduced pressure, and use of widely different times for electrotransfer. Special advantages with this method are low extent of protein α‐amino group destruction, direct use of the entire filter in the sequencer, and insensitivity to variations in electroblotting time. Gas‐phase hydrolysisin situof blotted proteins followed by amino acid analysis is known to give a low yield of polar amino acids, and often artifacts, but can still give an estimate of the amount of polypeptide immobilized on the filter. A wash withn‐butyl chloride is now shown to reduce the Polybrene‐associated artifacts, and an addition of sodium chloride to increase the recovery of polar amino acids. These two steps therefore appear interesting in schemes for compositional analyses of electro

ISSN:0173-0835    

DOI:10.1002/elps.1150110707

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

年代:1990

数据来源: WILEY

摘要:

AbstractSemidry electroblotting is convenient and allows a rapid and efficient protein transfer from one‐ or two‐dimensional polyacrylamide gels onto sequencer stable supports for protein microsequence analysis in a gas‐phase sequencer. Using this technique, we determined the amino acid sequences of the basic 7S globulin (Bg), an insoluble protein present in soybean seeds. Based on sequence determination, the cDNA‐encoding Bg could be easily cloned and charac

ISSN:0173-0835    

DOI:10.1002/elps.1150110708

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

年代:1990

数据来源: WILEY

摘要:

AbstractPurification and chemical characterization of protein may be achieved by combining two‐dimensional electrophoresis (2‐DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2‐DE pattern as possible. Under optimized conditions, blotting efficiencies between 60 % and 100 % were obtained for five marker proteins; the mean values from four regions of a 2‐DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60 % and 79 %. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patters, whereas single proteins occurred with higher amounts on either one or the other membrane. The results of the blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic p

ISSN:0173-0835    

DOI:10.1002/elps.1150110709

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

年代:1990

数据来源: WILEY

摘要:

AbstractRat liver glutathioneS‐transferases were partially purified usingS‐hexyl glutathione affinity chromatography, followed by native isoelectric focusing employing a pH 7–11 or pH 3–10 gradient. Proteins were excised and eluted from the gel for determination of subunit composition using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. In separate experiments, isoelectric focusing gels were equilibrated with a sodium dodecyl sulfate‐containing buffer at high pH, and proteins on the gel were electroblotted onto a polyvinylidene difluoride membrane, utilizing graphite plates as electrodes. The membrane‐bound proteins were visualized by Coomassie Brilliant Blue staining. The protein bands were then excised from the membrane and inserted into a gas phase sequenator for direct sequencing.N‐Terminal sequences thus determined were compared with published cDNA sequences. The isoelectric points (pIs) and positions on the isoelectric focusing gel of Yb1Yb1, Yb1Yb2and Yb2Yb2subunits were determined. We have also located on the pH 3–10 focusing gel anN‐terminal blocked glutathione S‐transferase which has a molecular weight s

ISSN:0173-0835    

DOI:10.1002/elps.1150110710

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

年代:1990

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150110711

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

年代:1990

数据来源: WILEY