摘要:

AbstractFluorescence studies carried out in solution indicate that in the presence of the anionic detergent sodium dodecyl sulfate (SDS) proteins are able to bind the noncovalent dye 1‐anilinonaphthalene‐8‐sulfonate (ANS). The interaction of this hydrophobic dye with protein‐SDS complexes is probably responsible for the fluorescence of protein bands observed by UV‐transillumination of polyacrylamide gels stained with ANS. It is shown that the staining of SDS‐polyacrylamide gels with ANS after electrophoresis allows the detection and quantitative estimation of proteins and peptides having different properties. The electrophoretic patterns obtained using ANS are equivalent to those obtained using Coomassie Brilliant Blue R‐250. The fluorescent staining method described in this work is simpler and much more rapid than conventional methods using

ISSN:0173-0835    

DOI:10.1002/elps.1150061102

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

年代:1985

数据来源: WILEY

摘要:

AbstractLinear polyacrylamide has been used to increase the tensile strength and stability of low percentage acrylamide gels in the range of 2.5 to 6.0 % w/v acrylamide. This added strength allows handling and staining of the gel without the excessive difficulty which is normally incurred when working in this range. Addition of up to 0.6 % w/v of linear polyacrylamide gives no pore size reduction. While addition of linear polyacrylamide to acrylamide gels using bisacrylamide as a crosslinker has a hazy appearance, the use of diallyltartardiamide as a crosslinker gives the desired clarity without loss of large pore size and improves adherence to glass walls. A further advantage of acrylamide gel matrices is that the traditional methods of silver staining can be used for detection of DNA. Thus, adding linear polyacrylamide to an acrylamide gel using diallytartardiamide as a crosslinker allows easy use of low percentage acrylamide gels for separation and detection of DNA fragments in the range of 70 to 2500 base pairs.

ISSN:0173-0835    

DOI:10.1002/elps.1150061103

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

年代:1985

数据来源: WILEY

摘要:

AbstractA thin‐layer counter current line absorption immunoelectrophoresis (TL‐CCLAIE) assay was developed for the analysis of antigens and antibodies to Aleutian disease virus. The TL‐CCLAIE assay is a rapid and sensitive assay employing 0.4 mm gels cast in moulds in order to allow technicians not routinely engaged in electrophoretic work to perform the assay. Thin gels allow a higher voltage and thus higher electrophoretic mobility of the antigens, making it possible to perform the complete assay withi

ISSN:0173-0835    

DOI:10.1002/elps.1150061104

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

年代:1985

数据来源: WILEY

摘要:

AbstractThe binding sites of non‐defatted and fatty acid‐free bovine serum albumin for bilirubin and biliverdin were investigated by means of analytical capillary isotachophoresis. In addition, the effect of oleic acid on the binding of bilirubin was examined with the same analytical tool. Albumin‐bilirubin complexes with increasing molar ratios of bilirubin can be identified as a separately migrating zone adjacent to the zone of free bilirubin. At pH 9 there are 3–4 high‐affinity binding sites for bilirubin and, possibly, several weaker binding sites. Fatty acid‐free and non‐defatted albumin exhibit almost identical binding properties with regard to bilirubin. Under the same conditions non‐defatted albumin shows only one high‐affinity binding site for biliverdin. The analysis of fatty acid‐free albumin‐biliverdin complexes reveals additional weak binding sites, which indicates that the physiological loading of albumin with ligands interferes only with the low‐affinity binding of biliverdin. At sufficiently high concentrations, oleic acid even competes for the first high‐affinity binding site of bovine serum albumin for bilirubin. There is a linear relationship between the zone length and the molar concentrations of free ligands (bilirubin, biliverdin). The differential binding characteristics of bilirubin and biliverdin indicate that the reduction of biliverdin yields a molecule which has a higher affinity for albumin; this, however, results in an inferior water solubility at physiological pH values. Owing to high resolution and quantification, capillary isotachophoresis is a powerful tool for screening endogenous and exogenous compounds with regard to their effect on the bilirubin tran

ISSN:0173-0835    

DOI:10.1002/elps.1150061105

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

年代:1985

数据来源: WILEY

摘要:

AbstractTwo methods which utilize simple buffers for the generation of stable pH gradients useful for preparative isoelectric focusing are compared and contrasted. The first employs preformed gradients comprised of two simple buffers in density‐stabilized free solution. The stability of this system is analyzed theoretically and by computer simulation. These precast gradients are limited to two buffering components, subject to diffusion, and restricted to the neutral pH region. An experimental application is presented. The second method utilizes neutral membranes to isolate electrolyte reservoirs of constant composition from the separation column. It is shown by computer simulation that steady state gradients can be formed at any pH range with any number of components in such a syste

ISSN:0173-0835    

DOI:10.1002/elps.1150061106

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

年代:1985

数据来源: WILEY

摘要:

AbstractThree common alleles are found in esterase D: EsD 1, EsD 2 and EsD 5. In addition, a number of rare variants are found. The isoeiectric points of the EsD 1 and EsD 2 isoenzymes differ by only 0.02 pH units, which could lead to possible confusion between the two using conventional isoelectric focusing (IEF). Therefore, in order to ensure adequate separation of these two bands, it is necessary to use a narrow pH gradient. The separation of esterase D (EsD) has been improved on electrofocusing gels by incorporating a narrow range Pharmalyte (pH 4.5–5.4) in conjunction with the separator N‐(2‐hydroxyethyl)piperazine‐N′‐2‐ethanesulphonic acid (HEPES). This mixture resulted in the formation of a narrow pH range of pH 4.85–5.35 (the major typing bands of EsD fall within the range of pI4.9–5.2). Resolution was further improved by the use of thickness modified pH gradients in ultrathin gels which flattened the pH gradient in the range of pH 5.0–5.1. The techniques used were shown to be effective for typing bloodstain

ISSN:0173-0835    

DOI:10.1002/elps.1150061107

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

年代:1985

数据来源: WILEY

摘要:

AbstractHuman factor B polymorphism has been investigated by isoelectric focusing in immobilized pH gradients in the range from pH 5.2 to 6.1, or 5.4 to 5.9, followed by nitrocellulose immunoblotting. Although the methodology offers a matchless mean to detect so far hidden factor B variability, we did not discover factor B subtypes while typing hundreds of fresh sera.

ISSN:0173-0835    

DOI:10.1002/elps.1150061108

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

年代:1985

数据来源: WILEY

摘要:

AbstractThe concentration of protein actually solubilized in sample buffer in preparation for analysis by two‐dimensional polyacrylamide gel electrophoresis cannot be directly determined by the Lowry, Biuret, or Bradford protein methods due to interference by the combinational effect of presence of urea, detergents, carrier ampholytes, and thiol compounds in sample solubilization buffers. Determinations of the actual amount of protein solubilized and applied to gels is required to accurately quantitatively and qualitatively evaluate second‐dimension polypeptide maps. It was found that when sample buffer consisting of 9 M urea, 4 % Nonidet P‐40, 2 % Ampholines, and 2 % 2‐mercaptoethanol containing solubilized sample(s) was acidified prior to dilution, protein concentrations over a range of 0.5 to 50 μg could be reproducibly determined utilizing a modified Bradford assay. The modified assay generates two near‐linear segments, one over the range<0.5 to 5 μg total protein that permits the application of Beer's law and a second linear response encompassing 5 to 50 μg total protein. The assay did not tolerate presence of greater than 0.1 % sodium dodecyl sulfate but addition of sodium chloride and protamine sulfate did not adversely affect protein quantitation. The modified assay allows direct quantitation of protein solubilized in sample buffers containing urea, carrier ampholytes, nonionic detergents, and thi

ISSN:0173-0835    

DOI:10.1002/elps.1150061109

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

年代:1985

数据来源: WILEY

摘要:

AbstractA metod is described for obtaining a sample‐stacking effect before sodium dodecyl sulfate‐agarose gel electrophoresis. The method requires no novel equipment and uses a discontinuous polyacrylamide‐agarose horizontal slab gel system. With the buffers selected, a complex mixture of proteins can be separated, according to the molecular weight of their subunits, in 15 % w/w agarose gels. The new gel system is versatile and may offer advantages over sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis in such applications as protein elution or enzyme renat

ISSN:0173-0835    

DOI:10.1002/elps.1150061110

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

年代:1985

数据来源: WILEY

摘要:

AbstractA new method is described for detecting inactive enzymes or other proteins after Cellogel electrophoresis. The proteins were blotted from the Cellogel strip to a nitro‐cellulose membrane and then the blot was blocked before incubation in a specific antibody solution. The antigen‐antibody complex was located by a second antibody staining procedure employing peroxidase‐linked goat anti‐rabbit IgG. The method described is sensitive and could be of general use for the detection of proteins after Cellogel electrop

ISSN:0173-0835    

DOI:10.1002/elps.1150061111

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

年代:1985

数据来源: WILEY