Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate—polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies

Ohhashi, Toshitaka; Moritani, Chie; Andoh, Hiromi; Satoh, Shuichi; Ohmori, Shinji; Lottspeich, Friedrich; Ikeda, Mikiko

doi:10.1016/0021-9673(91)85069-r

Cited by 24 publications

(13 citation statements)

References 14 publications

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“…The small recovery volume makes the samples easy to handle, and several samples should be eluted simultaneously to yield large amounts of protein. As other authors (Ohhashi et al, 1991), we could readily obtain milligram amounts of proteins with this method, enough to raise antibodies.…”

Section: Discussionmentioning

confidence: 61%

“…It is simple, fast, and cost-effective; it does not need an expensive apparatus and reagents, and does not require prior knowledge of the protein characteristics. This technique is applicable not only to soluble proteins but also to membrane-bound proteins and it is applicable to raise antibodies as well (Ohhashi et al, 1991). In addition, purification of proteins, particularly toxins, based on electroelution has been successfully implemented in previous studies (Borowiec et al, 2016; Hewlett et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

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A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin fromClostridium septicum

Vázquez-Iglesias

Estefanell-Ucha

Barcia-Castro

et al. 2017

PeerJ

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Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.

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Section: Discussionmentioning

confidence: 61%

Section: Discussionmentioning

confidence: 99%

A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin fromClostridium septicum

Vázquez-Iglesias

Estefanell-Ucha

Barcia-Castro

et al. 2017

PeerJ

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show abstract

“…Analytical and preparative slab gel electrophoresis methods have good separation resolution, but are limited by the small amount of material that can be separated [27]. Size exclusion HPLC is a fast method that can process large amounts of material, but the resolution of subunit separation is poor compared to that of gel electrophoresis.…”

Section: Discussionmentioning

confidence: 99%

A large scale preparative gel electrophoresis separation of α₁ and α₂ subunits of the voltage‐gated Ca²⁺ channel from rabbit skeletal muscle

1994

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A large-scale preparative gel electrophoresis method effectively separates individual voltage-gated calcium channel (VGCC) subunits with high resolution, starting with up to 4 mg of rabbit skeletal muscle L-type VGCC complex. Using this method, we separated alpha 1 and alpha 2 subunits of rabbit skeletal muscle VGCC with a high efficiency and with protein recoveries of 83%. The separated alpha 1 and alpha 2 subunits eluted from the gel in a 1:1 molar ratio. The method should be applicable for separating the other VGCC subunits or subunits of other protein complexes.

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“…(8)(9)(10) Protein band with a 26 kDa size was excised and cut into small fragments. Removing of CBB-R250 from the gel fragments was performed according to the Ball method.…”

Section: Removal Of Cbb-r250 and Electrophoretic Elution Of Proteins mentioning

confidence: 99%

“…The dialysis tubes were treated and electroelution was carried out as described previously. (10) Briefly, electroelution was performed at 50 V for 12 h at 48C in Tris-glycine buffer containing 0.1% SDS (pH 8.3). At the end of electrophoretic elution, the polarity of the electrodes was changed for 60 s in order to avoid the absorption of protein on the dialysis tubes.…”

Section: Removal Of Cbb-r250 and Electrophoretic Elution Of Proteins mentioning

confidence: 99%

Production of Polyclonal Antibody Against Alkyl Hydroperoxide Reductase ofHelicobacter pyloriand Its Antigenicity

et al. 2008

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Stool-antigen detection kits for diagnosis of Helicobacter pylori infection have been widely used because of their full non-invasive nature. Because Helicobacter pylori strains show a distinctive genetic diversity, it is important to find a protein that is a common antigen of various strains and shows a strong immunogenicity for the development of a stool- antigen detection kit. Alkyl hydroperoxide reductase (AhpC) of Helicobacter pylori strongly reacts with the sera of patients with gastritis and peptic ulcer. Therefore, AhpC seems to be an excellent candidate as a target protein for this study. Accordingly, polyclonal antiserum against AhpC was produced in adult New Zealand white rabbits by using AhpC in the gel bands without adding Freund's adjuvant. In addition, isolation and purification of AhpC were perfomed by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroelution. In this study, a simple method was used for rapid production of polyclonal antibody against AhpC of H. pylori, which avoids both the long-term AhpC purification and the addition of Freund's adjuvant. One-dimensional preparative gel electrophoresis allows a single and short purification step; the high-resolution capacity of this technique leads to a high level of purity of the protein and consequently to a very high specificity of the antibody. Moreover, this method avoids contamination by other non-specific proteins, which often appear during the purification process by column chromatographic techniques, which may also decrease the purity of the immunogen. The present method is simple, rapid and cost-effective, and also makes it possible to produce antibody for stool-antigen enzyme immunoassay in a short time and at low cost.

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Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate—polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies

Cited by 24 publications

References 14 publications

A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin fromClostridium septicum

A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin fromClostridium septicum

A large scale preparative gel electrophoresis separation of α₁ and α₂ subunits of the voltage‐gated Ca²⁺ channel from rabbit skeletal muscle

Production of Polyclonal Antibody Against Alkyl Hydroperoxide Reductase ofHelicobacter pyloriand Its Antigenicity

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Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate—polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies

Cited by 24 publications

References 14 publications

A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin fromClostridium septicum

A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin fromClostridium septicum

A large scale preparative gel electrophoresis separation of α1 and α2 subunits of the voltage‐gated Ca2+ channel from rabbit skeletal muscle

Production of Polyclonal Antibody Against Alkyl Hydroperoxide Reductase ofHelicobacter pyloriand Its Antigenicity

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A large scale preparative gel electrophoresis separation of α₁ and α₂ subunits of the voltage‐gated Ca²⁺ channel from rabbit skeletal muscle