摘要:

AbstractTwo‐dimensional electrophoresis with isoelectric focusing in immobilized pH gradients in the first dimension was applied to the fractionation of hydrophobic proteins from the plasma membrane of aStreptococcusstrain. The detergents incorporated into the first‐dimensional gel slab, especially the zwitterionic ones of the sulfobetaine series, interfere with protein transfer from the first to second dimension by formation of mixed micelles with sodium dodecyl sulfate molecules. In order to reduce their concentration an elution protocol was devised including: (i) fixation for 1 h in 50 % methanol ‐ 12 % acetic acid; (ii) washing with distilled water for 30 min; (iii) equilibration in concentrated Tris buffer; (iv) denaturation in 5 % sodium dodecyl sulfate ‐ 2 % 2‐mercaptoethanol. When counting the number of resolved spots in the protein patterns after two‐dimensional electrophoresis, the relative solubilizing efficiency of different detergents scored as follows: Nonidet P‐40 (NP‐40)>(3‐[3‐cholamidopropyl)dimethylammonio]‐1‐propananesulfonate (CHAPS)>N‐dodecyl‐N, N‐dimethylammonio‐3‐prop

ISSN:0173-0835    

DOI:10.1002/elps.1150071202

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

年代:1986

数据来源: WILEY

摘要:

AbstractProteins separated by isoelectric focusing of 20 samples and polyacrylamide gel electrophoresis of 10 samples in the same batch were stained with silver and were scanned with a computerized laser beam densitometer to obtain integrations values. A linear relationship was found between densitometric integration values (staining intensities) of proteins and their loads up to 600 ng (actin, carbonic anhydrase) or 1000 ng (albumin, creatine kinase), respectively. The same amount (250 ng) of applied albumin, actin, carbonic anhydrase and creatine kinase resulted in differences in staining intensities of these proteins; staining intensities of albumin and creatine kinase were similar, however: both were 1.8 X higher than the staining intensity of carbonic anhydrase and 2.3 X higher than of actin. Six human aortic intima proteins (ML‐1, ML‐2, P27, −14, P27,−18, P27,−19.5, P110) were selected for quantitative studies of the relationship between loads of intimal extract and the integration values of these proteins. Linear responses were found for these proteins in loads from 5 μL to 20 μL of intimal extract, corresponding approximately from 15 μg to 60 μg of total proteins applied to the focusing gel. When carbonic anhydrase was included with each sample, as an internal absorbance calibrator, we could express integration values of other proteins in relative amount in ng of carbonic anhydrase and thus compensate for variation in fixation, developing and staining conditions from b

ISSN:0173-0835    

DOI:10.1002/elps.1150071203

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

年代:1986

数据来源: WILEY

摘要:

AbstractA computer‐controlled version of recycling isoelectric focusing has been applied to the purification of recombinant human leukocyte interferons (IFNs) fromE. coli. The system is based on continuous recycling between a 12‐channel heat‐exchange reservoir and a 12‐channel focusing cell. It contains ten pH meters with probes in each of the flowing channels and two ultraviolet spectrophotometers with five flow cells per instrument. A minicomputer system acquires real‐time data of pH and optical density measurements, controls and continuously monitors voltage and power outputs across the focusing cell, and generates on‐line multi‐color graphics on anx‐yplotter. Recycling isoelectric focusing in the presence of 4 M urea resulted in removal of contaminating bacterial pyrogens from recombinant IFN alpha‐2 with complete retention of specific antiviral activity. It also produced a 25‐fold purification of crude recombinant IFN alpha‐5 in a single step and resolved two biologically active species. These data suggest that recycling isoelectric focusing may be incorporated into the design of simple schemes for the purification of a variety of

ISSN:0173-0835    

DOI:10.1002/elps.1150071204

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe theoretical principle and experimental verification of a new separation method for proteins in thin gel slabs called “isotachophoretic focusing” are presented. Isotachophoretic focusing is basically isotachophoresis with two new features: (i) By using adequate spacers a continuous mobility spectrum is generated. Under isotachophoresis conditions, these spacers give rise to a linear electric field gradient in which proteins can be separated in form of focusing bands. The synthesis and isolation of suitable spacers for isotachophoretic focusing are described. (ii) The stack is immobilized or, at least, its migration is retarded by exploiting the counterflow of electroendosmosis resulting from the action of the electric field on charged groups linked to the gel. Immobilization of the stack allows its formation to equilibrium and the protein separation process to continue as long as necessary. Details are given concerning the experimental set‐up and factors involved in the optimization of the technique. The separations obtained by isotachophoretic focusing are compared with those obtained by classical electropho

ISSN:0173-0835    

DOI:10.1002/elps.1150071205

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

年代:1986

数据来源: WILEY

摘要:

AbstractAmpholine pH 4–6 and Pharmalyte pH 4.2–4.9, 4.5–5.4 or 5–6 have been used to improve the resolution of the simplified technique of high resolution two‐dimensional electrophoresis. All give excellent resolution but Pharmalyte interferes with protein detection methods to an extent which increases with the pH range of the mixture. The effect is characterised by a progressive increase in the intensity and area of background stain associated with the anode and cathode regions of the two‐dimensional gels. It is minimal following Serva Blue R (Coomassie Blue) staining but severe following silv

ISSN:0173-0835    

DOI:10.1002/elps.1150071206

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

年代:1986

数据来源: WILEY

摘要:

AbstractDirect gene analysis of the haptogobin (Hp) polymorphism was carried out by Southern blotting using an Hp cDNA probe. Two types of polymorphism were observed: the first, afterEcoRI,HindIII andPstI digestion, is due to an intragenic duplication and corresponds to the Hp‐α protein polymorphism; the second type, due to point mutation, was represented by two additional restriction sites forEcoRI andPstI. The univocal correspondence between the first type fragment length variants and protein polymorphism of the duplication type permits the establishment of the genotype at the DNA level, and mutational variants of the second type add new markers; the method described here also permits detection of the real genotype of Hp0 allel

ISSN:0173-0835    

DOI:10.1002/elps.1150071207

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

年代:1986

数据来源: WILEY

摘要:

AbstractFree‐flow electrophoresis of proteins in solutions of borax and mannitol was investigated. The borax solution represents an equimolar boric acid‐NaOH buffer with a pH of 9.2. On addition of mannitol the pH of the buffer decreases, with increasing polyol concentration, accompanied by a slight decrease in conductivity. Using a 2.5 mM borax solution, buffers with pH from 9.2 to 4.5 can be obtained. Under the influence of the electric field the pH and conductivities of these buffers change in the near‐membrane regions of the separation chamber. Despite the changes the buffers are suitable for electrophoresis of proteins in a free‐flow apparatus. The separation of bovine hemoglobin as well as a mixture of bovine hemoglobin, human serum albumin and cytochrome C in borax‐mannitol solutions is demonstrated at 80–100 mA constant current and a residence time of only

ISSN:0173-0835    

DOI:10.1002/elps.1150071208

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

年代:1986

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150071210

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

年代:1986

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150071211

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

年代:1986

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150071212

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

年代:1986

数据来源: WILEY