摘要:

AbstractWhen using agarose gel electrophoresis to fractionate linear DNA longer than 40–60 kilobases (kB), improved resolution by length has previously been obtained by periodically and discontinuously changing the direction the electrical field. This change of the field's direction was accomplished by alternately activating two sets of electrodes. However, some of the procedures used produce inhomogeneous electrical fields and with all of these procedures it is comparatively difficult to either change the angle between the two directions of the field or suppress formation of local pH gradients. To overcome these problems, a procedure is presented for changing the direction of the electrical field by rotating the gel. During agarose gel electrophoresis, a commercially available stepping motor and indexer are used to periodically rotate a gel on a circular disk within a conventional, horizontal, submerged gel electrophoresis apparatus. Because of the homogeneous field used, DNA forms comparatively sharp, undistorted bands. The previously achieved improved resolution of 40–166 kB, linear, double‐stranded DNÀ has been achieved here by rotating the gel. Dependence of length resolution on both the angle of rotation and the temperature has been me

ISSN:0173-0835    

DOI:10.1002/elps.1150080702

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

年代:1987

数据来源: WILEY

摘要:

AbstractThe mechanism by which three basic Immobilines (pK's 6.2, 7.0 and 9.3) precipitate proteins in solution seems to be due to the presence in these three monomers of variable amounts of oligomers, spontaneously formed during storage by polymerization. These polymerization products range in size from short oligomers (dimers and trimers) to polymers with a molecular mass more than 2000 Da. Oligomers are present in all batches of precipitating Immobilines in substantial amounts, usually at least 15–20%. Only the pK8.5 Immobiline, in general unable to precipitate proteins from solution, has been found to contain low levèls of oligomers, typically less than 3%, possibly due to intrinsic stability against polymerization, or to its ability to spontaneously hydrolyze in water solutions. None of the batches of pK6.2, 7.0 and 9.3 Immobilines, unable to precipitate proteins in solution, was found to contain polymers but exhibited a bimodal elution peak from a Bio‐Gel P‐2 sieve gel chromatography, probably corresponding to dimers and trimers. Upon mild polymerization (highly diluted persulfate in presence of oxygen) all these batches were found to precipitate proteins and to contain higher oligomers and polymers. By testing column eluates, it was found that the precipitation power begins at the level of polymers with 10–12 residues, suggesting that these relatively short chains already possess a strong cooperativity in their chemical behavior in solution. Work is in progress to find a suitable solution for completely inhibiting two of the most noxious reactions occurring to Immobilines: hydrolysis and polymerization. There still remains a third problem, which will require a longer‐term investigation: the intrinsic hydrophobicity of the four basic Immobilines. The basic Immobiline homopolymers present two unlike groups for interaction: ionic on one side, hydrophobic on the opposite end. Acidic Immobilines, even when reacted to form much larger polymers, are unable to precipitate ferritin from solution, suggesting that their strongly hydrophilic surface suppresses one of the two binding regions. In a similar manner, when batches of pK7.0 Immobiline, able to precipitate ferritin, where rendered more hydrophilic by methylolation, their protein precipitation power was abolished, even, though the polymers had not been eliminated. While waiting for the synthesis of more ‘hydrophilic’ Immobilines, a partial remedy is to admix carrier ampholytes (CA) to Immobiline gels. The CA's act as shielding ions, minimizing hydrophobic interactions among proteins and the Immobiline matrix. Mixed CA‐IEF gels should never be prefocused, as the shielding action of CA is maximal in the

ISSN:0173-0835    

DOI:10.1002/elps.1150080703

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

年代:1987

数据来源: WILEY

摘要:

AbstractAffinity electrophoresis was applied to the study of binding of monoclonal antibodies (MoAb's) with their respective antigens. The MoAb's may be either mixed with the antigen in an application well or incorporated into the agarose gel. The first approach is more convenient for studying high affinity MoAb's, while MoAb's with low affinity might be more efficiently studied in the second system. Our conditions for affinity electrophoresis do not imply nor necessitate immobilization of either an antigen or MoAb and are most suitable for studies of MoAb's reacting with antigens of alpha mobility demonstrated in experiments with MoAb's against human alpha‐fetoprotein (AFP). Both AFP‐MoAb and AFP‐MoAb‐AFP complexes were observed during electrophoresis. The identity of these complexes was confirmed by their reaction both with polyclonal antibodies against mouse immunoglobulins and against human AFP. The molecular weight of complexes was determined by gel filtration. Affinity electrophoresis was less convenient for studies of MoAb's reacting with antigens of gamma mobility at pH 8.6. Nevertheless, MoAb's against carcinomembryonic antigen, human IgG and human IgA did bind with their respective antigens under the conditions of affinity electrophoresis. The binding of MoAb's with these antigens during affinity electrophoresis was confirmed by demonstration of mouse immunoglobulins in the precipitates formed by reaction of the studied antigens with polyclonal antibodies. Our results point out the suitability of affinity electrophoresis for studies of

ISSN:0173-0835    

DOI:10.1002/elps.1150080704

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

年代:1987

数据来源: WILEY

摘要:

AbstractBovine salivary proteins have been shown to be correlated with bloat susceptibility in cattle. Due to the presence of the high molecular weight mucoprotein in whole saliva these proteins have been difficult to purify. This difficulty has now been overcome by using electrophoretic procedures. The purification protocol consisted of electrophoretic concentration of the saliva, separation of the proteins by polyacrylamide gel electrophoresis, excision of the protein bands followed by electroelution and concentration. Purified proteins were obtained from bands 4, 5, 6, 7, 8 and 9/10. The molecular weights of the intact proteins were between 40 300 and 112 400 on gel chromatography and the subunit molecular weights were between 15000 and 65 400. The isoelectric points were within the pH range of 3.6 to 5.1. Antisera were produced against protein bands 7 and 8 and these were used to determine antigenic relationships between proteins and as a means of quantifying the proteins by the Laurell rocket immunoassay.

ISSN:0173-0835    

DOI:10.1002/elps.1150080705

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

年代:1987

数据来源: WILEY

摘要:

AbstractLipoproteins in human serum and in lipoprotein fractions obtained by sequential ultracentrifugation of serum were analyzed by combining three types of micro two‐dimensional electrophoresis (2‐DE) technique to obtain systematic information on lipoproteins and apolipoproteins. The samples were first analyzed under non‐denaturing conditions of electrophoresis to characterize the physicochemical properties of low‐density lipoproteins (LDL) and high‐density lipoproteins (HDL) under physiological conditions. The samples were then analyzed, employing isoelectric focusing without denaturants in the first dimension and sodium dodecyl sulfate (SDS) electrophoresis in the second dimension, to study the process of dissociation of lipoproteins to apolipoproteins. The HDL's dissociated into their constituent apolipoproteins, showing that the charge heterogeneity of HDL is caused by the heterogeneous apolipoprotein content. LDL dissociated into apo B‐100 and minor components. In the third type of micro 2‐DE, employing urea and Nonidet P‐40 in the first dimension and SDS in the second dimension, the lipoproteins were dissociated into their apolipoproteins during the steps of sample treatment, to obtain the information on the contnt of apolipoprotein peptides and their genetic alleles. The combined micro 2‐DE technique will be useful for the systematic analysis of lipoproteins and apolipoproteins in hypo‐ and hyperlipop

ISSN:0173-0835    

DOI:10.1002/elps.1150080706

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

年代:1987

数据来源: WILEY

摘要:

AbstractWe have examined the utility of tetramethylurea as a protein denaturant in electrophoresis using transverse gradient gels. Its physical properties make it easier to work with than urea and it appears to denature bovine serum albumin in a similar way. Tetramethylurea is a potential substitute for urea in some electrophoretic systems.

ISSN:0173-0835    

DOI:10.1002/elps.1150080707

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

年代:1987

数据来源: WILEY

摘要:

AbstractA new method of identifying allo A lectin‐binding glycoproteins separated by isoelectric focusing is described. The method is based on the combination of neuraminidase treatment, isoelectric focusing and Durapore filter blotting followed by detection with allo A lectin. The technique is simple and reproducible for the determination of both the alpha 1‐antitrypsin and orosomucoid phenotypes in genetic and forensic investigati

ISSN:0173-0835    

DOI:10.1002/elps.1150080708

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

年代:1987

数据来源: WILEY

摘要:

AbstractA new method is presented for the fast preparative separation of the light‐harvesting photosynthetic pigment C‐phycocanin into its α and β subunits, which is based on isoelectric focusing in layers of granutaled gels containing 7 M urea. The method has been successful in cases where other separation procedures failed. The recovery of the separated chains of the light ‐sensitive biliprotein amounts to 70 ± 10 % when the separation is carried out under light exclusion and in an argon atmosphere. A simple and inexpensive setup for work under an atmosphere of protective gas is d

ISSN:0173-0835    

DOI:10.1002/elps.1150080709

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

年代:1987

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150080710

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

年代:1987

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150080711

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

年代:1987

数据来源: WILEY