Chemistry: analytical and immunological testing – Peptide – protein or amino acid
Reexamination Certificate
1999-07-10
2001-08-21
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
C436S088000, C436S164000, C436S166000, C436S169000, C436S174000, C008S636000, C008S657000, C008S658000
Reexamination Certificate
active
06277643
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for detection of protein on polyacrylamide gels using a counter-dye composition and the counter-dye composition for detection of protein on polyacrylamide gels. More specifically, the present invention relates to a method for detection of protein in a high sensitivity on polyacrylamide gels in a rapid and simple manner, comprising the step of staining polyacrylamide gels with a counter-dye composition containing an acidic organic dye and a basic organic dye, and the counter-dye composition for detection of protein on polyacrylamide gels.
BACKGROUND ART
Polyacrylamide gel electrophoresis (PAGE) is a protein analysis technique which is useful in the separation, identification, approval of purity and determination of size of protein. Particularly, SDS-PAGE (Sodium Dodesyl Sulfate-PAGE) is a method characterized in that protein binds intimately with an anionic surfactant, i. e. SDS, with the ratio of 1.4 to 1, so as to have (−) charge on the surface thereof, and thereby only the size thereof acts as a separation factor. This method is widely used in protein analysis because it can be simply-handled and has a good resolution (see [A.T. Andrews, Electrophoresis, 2nd Ed., Oxford Science Publications, 1-58, (1988)])
Since most of proteins, which are subjected to analysis, are colorless, an appropriate method must be considered for detection. Various detection methods have been reported to the present time such as organic dye staining method, silver staining method, fluorescence staining method and background staining method, etc.
Organic dye staining method is performed by staining protein band with an organic dye such as Amido black 10B, Ponceaus S, Fast green FCF, Coomassie brilliant blue R (abbreviated to CBBR), etc (see [J. of Chromatography A, 698, 123-143 (1995)]). Particularly, since CBBR staining method is relatively simple and inexpensive, it has been generally used. However, it has problems in requiring a long time staining and destaining procedure and not having so good sensitivity. (50 ng on Bovine serum albumin (BSA)).
Silver staining method is performed by adsorbing silver to gels and then, carrying out reduction reaction, which has the highest sensitivity among non-isotopic detection methods. Although its sensitivity reaches to 0.1 ng, it has problems in involving difficult and multiple steps.
Fluorescence staining method is performed by labelling protein with a fluorescent dye. Although it has high detection sensitivity, it has drawbacks in that it involves very complicated steps, and requires the radiation of ultraviolet rays and the use of expensive equipment for quantitation.
Background staining method is performed by forming precipitate on gels except for protein band, the use of which is limited to SDS gels. Although this method is rapid and useful, the resulting band is not persistent and it has difficulty in storing the stained gel. (see [Anal. Biochem. 174, 157-167 (1988)])
Under the above-described situation, it has been required to develop a novel method for detection of protein in a higher sensitivity than the prior method on polyacrylamide gels in a rapid and simple manner.
DISCLOSURE OF THE INVENTION
Accordingly, the purpose of the present invention is to provide a method which can solve the problems involved in the above-mentioned prior methods. Thus, the present invention relates to a method for detecting protein in a high sensitivity, and in a rapid and simple manner, by staining polyacrylamide gels with a counter-dye composition.
One aspect of the present invention provides a method for detection of protein on polyaciylamide gels comprising the step of staining the polyaciylamide gels with a counter-dye composition containing an acidic organic dye and a basic organic dye. Preferably, the composition contains Zincon and Ethyl violet, Zincon and Methyl violet, Zincon and Meldola's blue, Coomassie brilliant blue R and Phenosafranin, Coomassie brilliant blue G and Methyl orange, Calconcarboxylic acid and Rhodamine B, or Eriochrome black T and Rhodamine B. More preferably, the composition contains Zincon and Ethyl violet, Zincon and Methyl violet, Coomassie brilliant blue R and Phenosafranin, or Coomassie brilliant blue G and Methyl orange. Most preferably, the composition contains Zincon and Ethyl violet, or Zincon and Methyl violet. In the present composition, preferably, Zincon has a concentration of 0.001-0.02% by weight of the volume of the composition. Preferably, the molar ratio of Zincon to Methyl violet or Ethyl violet is 1 to 0.8 and the composition further contains 7 v/v % acetic acid-containing 35 v/v % aqueous methanol solution.
Another aspect of the present invention provides a counter-dye composition for detection of protein on polyacrylamide gels containing an acidic organic dye and a basic organic dye. Preferably, the composition contains Zincon and Ethyl violet, Zincon and Methyl violet, Zincon and Meldola's blue, Coomassie brilliant blue R and Phenosafranin, Coomassie brilliant blue G and Methyl orange, Calconcarboxylic acid and Rhodamine B, or Eriochrome black T and Rhodamine B. More preferably, the composition contains Zincon and Ethyl violet, Zincon and Methyl violet, Coomassie brilliant blue R and Phenosafranin, or Coomassie brilliant blue G and Methyl orange. Most preferably, the composition contains Zincon and Ethyl violet, or Zincon and Methyl violet. In the present composition, preferably, Zincon has a concentration of 0.001-0.02% by weight of the volume of the composition. Preferably, the molar ratio of Zincon to Methyl violet or Ethyl violet is 1 to 0.8 and the composition further contains 7 v/v % acetic acid-containing 35 v/v % aqueous methanol solution.
REFERENCES:
patent: 4966854 (1990-10-01), Fleming
patent: 5705649 (1998-01-01), Shultz et al.
Choi, et al. A Modified Coomassie Blue Staining of Proteins in Polyacrylamide Gels with Bismark Brown R, Anal. Biochem. 236(1):82-4 (Apr. 5, 1996).
Choi Jung Kap
Jung Da-Woon
Tak Gyoung Hoon
Yoo Gyurng Soo
Choi Jung Kap
Corless Peter F.
Dike Bronstein, Roberts & Cushman LLP
Warden Jill
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