Procedure to be performed in conjunction with protein blotting o

Details Procedure to be performed in conjunction with protein blotting o Procedure to be performed in conjunction with protein blotting o

1987-05-27

1989-05-02

Marantz, Sidney

Chemistry: analytical and immunological testing

Automated chemical analysis

Utilizing a centrifuge or compartmented rotor

436530, 436507, 435 6, 435310, 435312, G01N 3500, C12Q 168, C12M 110

Patent

active

048267716

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

The present invention relates to a simplified procedure to be performed in conjunction with protein blotting or nucleic acid blotting, in that the medium to which said components have been transferred is contacted with reagent and washing solutions in a rotary drum.
When an electrophoretic separation is carried out the sample solution is usually applied onto a gel disk or plate having a thickness of about one millimeter or a few millimeters, whereupon the individual components of the sample are caused to migrate in the gel under the action of an electric field. For an identification and analysis of the separated components and, optionally, for treating them with suitable reagents it is imperative that the whole amount of the component contemplated is made available. Since in a so-called electrophoretic band the components are in a diffused state such as to extend into the interior of the gel--that is, they are distributed within the volume defined by the surface area of the band and the thickness of the gel--methods have been developed by which component molecules are transferred from the gel to the surface of a solid matrix, usually nitrocellulose paper. Such a transfer is generally referred to as "blotting". A blotted protein by being located on a surface rather than inside a gel is enabled to participate in a major number of interactions. Thus, for example, it may react with antibodies; this is an important feature for immunological purposes. Some prominent examples of such practical applications are specific antisera.
Antibodies bound to blotted proteins can be detected in various ways. A method frequently chosen involves the use of a labeled secondary antibody directed against the primary antibodies. Such a secondary antibody may be for instance antirabbit-IgG in cases where the primary antibody source is rabbit serum. Labeling of the antibody may be effected with the aid of some radioactive isotope, fluorescent compounds or certain types of enzymes. In this latter case the enzyme position on the solid matrix is stained by after-treatment with suitable substrates. An increased degree of sensitivity may sometimes be obtained by the use of a secondary antibody which is allowed to react with a labeled reagent in a third step. Recent techniques employ for example biotin-labeled secondary antibodies which will react in a third step with the protein avidin; this has multiple binding sites for biotin and has attached to it an enzyme as for instance, in most cases, either alkaline phosphatase or peroxidase. With this technique it is possible to detect amounts of protein down to about 100-200 picograms.
From the above explanations it will be appreciated that the protein that has been transferred to a solid matrix by way of blotting will be subjected to a major number of treating steps in connection with reactions of the kinds indicated above. Such treatments thus may involve three or more reagent solutions plus intercalated washing steps. This procedure is carried out, according to current techniques, in that the matrix containing the blotted proteins is incubated in boxes or sealable plastic bags on various types of shaker or rocker devices, the reagents/wash liquors being exchanged after predetermined periods of times. Frequently a plurality of samples are analyzed in parallel tests in which one or more of the treating steps may be the same. In cases where the protein is to be treated with three reagent solutions and three rinses in succession this will be found to involve a sequence of twelve treating steps; it will be readily appreciated that this is a cumbersome and lengthy procedure, with present techniques. Note moreover that a certain minimum amount of reagent is required for carrying out an effective incubation; and in order to suit the types of containers currently employed this minimum amount unfortunately has to be a relatively large volume. This is a significant disadvantage since some reagents are very expensive and may be available in only minute amounts - as e.g. ce

REFERENCES:
patent: 3626834 (1969-09-01), Speranza
patent: 3950134 (1976-04-01), Miles
patent: 4302092 (1981-11-01), Ashton et al.

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