Chemistry: analytical and immunological testing – Peptide – protein or amino acid
Reexamination Certificate
2001-08-03
2003-04-29
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
C436S015000, C436S164000
Reexamination Certificate
active
06555382
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the field of detection of proteins using synthetic dyes and, more particularly, to the detection of proteins in electrophoretic gels.
2. Description of the Related Art
One of the most commonly used and valuable methods for the separation and analysis of proteins is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this method, a sample containing protein is separated by electrophoresis in a sodium dodecyl sulfate-containing buffer through a polyacrylamide gel. As proteins are generally colorless, the gel is usually stained after electrophoresis to visualize the proteins.
A variety of methods are available for staining the proteins in such a gel. All of these methods, however, suffer from disadvantages that are addressed by the present invention.
For example, U.S. Pat. No. 4,555,490 (Merril) entitled “Rapid Visualization System for Gel Electrophoresis” describes a photodevelopment method involving silver ions. The procedure involves fixing the gel with methanol/acetic acid/citric acid solution, and using a methanolic silver nitrate solution. Methanol/acetic acid solutions have a considerable odor. This fixing solution is apparently essential prior to silver staining. Particular care must also generally be taken to avoid contact with silver nitrate solutions. Moreover, in addition to the contact and disposal problems associated with the use of silver ion, some care must apparently be taken to prevent silver salts from precipitating on the surface of the gel.
A number of methods use one of the dyes known as COOMASSIE brilliant blue to stain proteins. For example, U.S. Pat. No. 4,219,337 (Grossberg et al.) entitled “Assay for Proteins and Polypeptides” describes the use of COOMASSIE brilliant blue G250 in perchloric or hydrochloric acid, which couples with the protein and undergoes a color change. This patent does not describe the use of this reagent to stain proteins in polyacrylamide gels.
U.S. Pat. No. 4,946,794 (Berube) entitled “Visualization of Proteins on Electrophoresis Gels Using Planar Dyes” describes the use of COOMASSIE brilliant blue R250 or other dyes to stain proteins in polyacrylamide gels. The procedure requires a staining step in a methanol/acetic acid solution of the dye for one hour and a 15 minute potassium dichromate treatment to complex the dye and differentiate between the polyacrylamide matrix and the protein-containing spot. Thus, this procedure also requires the unpleasant use of methanol/acetic acid, and requires a one hour staining step.
U.S. Pat. No. 5,273,906 (Shultz et al.) entitled “Protein Staining Compositions and Methods” describes a series of derivatives of COOMASSIE dyes which are designed to overcome some of the problems usually associated with the use of COOMASSIE dyes in SDS-PAGE gels. However, in staining gels with these dyes, the staining solution is a methanolic solution, involving the problems associated with handling and disposal of methanol. Moreover, COOMASSIE brilliant blue dyes are more widely available and inexpensive than the derivatized COOMASSIE dyes of the Shultz patent.
In addition to the aforementioned patents, an article by Nivinskas et al. (Bio Techniques 20:380-385, 1996) entitled “Environmentally Benign Staining Procedure for Electrophoresis Gels Using COOMASSIE Brilliant Blue” describes a procedure for staining gels with dilute aqueous solutions of COOMASSIE brilliant blue R. The SDS-PAGE gel was typically first rinsed and washed in a large volume of dilute HCl for two hours. The gel could be boiled in water, then rinsed, but this produced no difference in staining. The staining step involved overnight staining by a 0.0015% (w/v) solution of COOMASSIE brilliant blue G-250 in 1 mM HCl. Bands begin to appear after about one hour but the gel must be stained overnight (i.e., 16 hours) for maximal staining and quantitation, then destained with a large volume of 1 mM HCl and absorbent tissue wipes to absorb unbound dye. Although this method eliminates the problems associated with methanol and acetic acid, it is still slow and requires several solution changes.
Thus, I have noted that the existing methods of staining gels suffer either from problems of use of undesirable reagents, such as acetic acid and methanol or silver nitrate, or from long processing times. Additionally, I have noticed that some of the methods are inconsistent in the staining results, which can lead to the ruining of an experiment. Based on my reading of the contemporary art, I have determined that what is needed is a faster and easier way of staining proteins in and/or on solid matrices and supports.
SUMMARY OF THE INVENTION
It is therefore an object of my invention to provide an improved method of staining proteins embedded in and/or on a solid matrix (e.g., a gel or porous particle) or solid support (e.g., a membrane or porous filter).
It is also an object of the invention to provide a method of staining proteins which takes only a very short time (i.e., less than about one hour, 30 minutes, or 15 minutes).
It is a further object of the invention to provide a method of staining proteins which uses aqueous solutions.
It is a yet further object of the invention to avoid the use of certain noxious alcohols (e.g., methanol) and/or organic acids (e.g., acetic acid) as the solvent of the solutions being utilized, or a major component thereof.
It is a still further object of the invention to provide a staining method which consistently and reproducibly stains proteins.
It is a still further object of the invention to avoid the use of a special fixing step (e.g., acetic acid as a fixative) before protein staining.
These objects as well as other which will be apparent from the description below are achieved by the invention which provides a method for staining immobilized proteins (e.g., immobilized in a polyacrylamide slab gel, in an agarose or polyacrylamide bead, on and/or in a flexible membrane, on and/or in a disc or fiber filter) using at least one or two brilliant blue stains. This method may include the steps of: washing the immobilized protein(s) on and/or in a solid matrix or support in hot wash solution, staining the immobilized protein(s) in a hot staining solution containing brilliant blue dye in dilute acid, and rinsing the solid matrix or support to rinse the dye which is not associated with the immobilized protein(s). Washing and destaining steps may be considered as optional for performing the method of the invention, but they may improve the results obtained.
The wash solution may be water or a dilute acid (e.g., hydrochloric or perchloric acid). By “hot” is meant a solution heated to substantially above room temperature and the solution may cool thereafter or be maintained at the initial temperature. The hot wash solution may initially be at a temperature greater than 45° C., greater than 90° C., or brought to a boil. The washing step may be achieved by placing the gel in the wash solution and then removing the solution at the end of the incubation period. The wash solution may be heated in a microwave oven or on a hot plate, before or after the gel is placed therein, until the desired temperature is achieved.
Likewise, the staining solution is heated to substantially above room temperature; the hot staining solution may initially be at a temperature greater than about 45° C., greater than about 70° C., greater than about 90° C., or brought to a boil. The staining step may be achieved by placing the gel in the staining solution and then removing the solution at the end of the incubation period. The staining solution may be heated in a microwave oven or on a hot plate, before or after the gel is placed therein, until the desired temperature is achieved.
For a solid support (e.g., a membrane or filter), the entire procedure may be done at room temperature. Here, washing might not be required. But, as in the applications involving a solid matrix, extended rinsing (e.g., long incubation time, agitation, multiple changes of rinsing soluti
Cole Monique T.
Warden Jill
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