News Column

Patent Application Titled "Modified Electrode Buffers for Stain-Free Protein Detection in Electrophoresis" Published Online

March 4, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventor Belisle, Christopher (Walnut Creek, CA), filed on August 7, 2013, was made available online on February 20, 2014 (see also Bio-Rad Laboratories, Inc.).

The assignee for this patent application is Bio-Rad Laboratories, Inc.

Reporters obtained the following quote from the background information supplied by the inventors: "Gel electrophoresis, most notably in polyacrylamide gels, is one of the most common laboratory techniques for analyzing biological samples for their protein contents, including both identification and quantitation. Detections of proteins in gels are achieved in a variety of ways. The most common is the use of stains such as COOMASSIE.TM. Brilliant Blue (BASF Aktiengesellschaft, Ludwigshafen, Germany), Ponceau S (Sigma-Aldrich, St. Louis, Mo., USA), and SYPRO RUBY.TM. (Life Technologies). Detection can also be achieved without stains, such as by using a stain-free technique disclosed by Edwards et al. in U.S. Pat. Nos. 7,569,103 B2 (Aug. 4, 2009) and 8,007,646 B2 (Aug. 30, 2011). These patents describe the UV light-induced reaction between the indole moiety of tryptophan and any of various halo-substituted organic compounds to produce a fluorescent derivative of the protein that emits light at wavelengths in the near-ultraviolet and visible range. The stain-free technique thus entails contacting the proteins or the gel with the halogen-containing reagent, exposing the gel to UV light once the proteins have been separated in bands within the gel by electrophoresis, and detecting, or forming an image of, the emissions from the proteins."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventor's summary information for this patent application: "It has now been discovered that a halo-substituted organic compound that reacts with tryptophan residues can be distributed through a gel for reaction with proteins in the gel by imposing an electric charge on the compound and incorporating the charged compound in one or both of the electrode buffers in an electrophoresis system. When a biological sample is loaded onto the gel and the electrodes that are immersed in the buffers are energized at the appropriate polarities to cause electrophoretic separation of the proteins in the sample to occur, the halo-substituted compound will migrate into and through the gel by virtue of its charge, thereby utilizing the electrophoretic principle to transfer the compound from the electrode buffer into the gel. The penetration of the gel with the halo-substituted compound will thus occur concurrently with the reparatory migration of the proteins within the gel, avoiding any need for pre-treatment of the sample or the gel or for post-treatment of the gel.

"A method is provided herein for separating proteins contained within a biological sample by electrophoresis in a gel along a linear dimension between first and second ends of the gel and detecting the proteins so separated. The method includes the steps of: (a) loading the sample onto the gel and electrically connecting the first and second ends of the gel with electrodes through electrode buffers in which the electrodes are immersed, wherein at least one of the electrode buffers has suspended therein a halo-substituted organic compound that reacts with tryptophan residues upon irradiation with ultraviolet light to form fluorescent compounds, the halo-substituted organic compound bearing an electrical charge, (b) energizing the electrodes to opposing polarities in a direction selected to cause the proteins to distribute within the gel along the linear dimension and to cause the halo-substituted organic compound to migrate into the gel from the electrode buffer, and with the proteins so distributed and the halo-substituted organic compound having so migrated into the gel, irradiating the gel with ultraviolet light to react tryptophan residues on the proteins with the halo-substituted organic compound to form fluorescent derivatives of the proteins and detecting fluorescent signals emitted from said fluorescent derivatives.

"In some embodiments of the method, the halo-substituted organic compound bears a negative charge and step (b) includes energizing the electrode that is immersed in the electrode buffer in which the halo-substituted organic compound is suspended as a cathode. In one such embodiment, the halo-substituted organic compound has a molecular structure that contains a negative ionic moiety. In other such embodiments, the halo-substituted organic compound is a hydrophobic compound encapsulated in a negatively charged micelle. In one of these embodiments, the micelle is formed of sodium dodecyl sulfate.

"In some embodiments of the method, the halo-substituted organic compound bears a positive charge and step (b) includes energizing the electrode that is immersed in the electrode buffer in which said halo-substituted organic compound is suspended as an anode. In some embodiments, the electrodes at the first and second ends of the gel are immersed in the same electrode buffer.

"Also provided herein is a method of detecting proteins in an electrophoresis gel. The method includes the steps of: loading the proteins into the electrophoresis gel; placing the electrophoresis gel between two electrodes; contacting the electrophoresis gel and one or both electrodes with an electrode buffer, the electrode buffer containing a halo-substituted organic compound; energizing the electrodes to opposite polarities, thereby causing migration of proteins within the gel and transfer of the halo-substituted organic compound from the electrode buffer into the electrophoresis gel; exposing the electrophoresis gel to ultraviolet light; and detecting fluorescence emitted from the electrophoresis gel, thereby detecting proteins in the electrophoresis gel.

"Some embodiments of the present methods also involve transferring proteins from the electrophoresis gel to a blotting membrane using electroblotting; exposing the blotting membrane to ultraviolet light; and detecting fluorescence emitted from the blotting membrane, thereby detecting proteins on the blotting membrane.

BRIEF DESCRIPTIONS OF THE DRAWINGS

"FIG. 1 shows fluorescence emitted by proteins in Gel A of the Example. Gel A contained 0.5% trichloroethanol, which was incorporated into the gel upon pouring.

"FIG. 2 shows fluorescence emitted by proteins in Gel B of the Example. No halo-substituted organic compound was incorporated into the gel upon pouring, but electrophoresis was carried out in the presence of an electrode buffer containing 0.5% trichloroethanol.

"FIG. 3 shows fluorescence emitted by proteins in Gel C of the Example. No halo-substituted organic compound was incorporated into the gel upon pouring, but electrophoresis was carried out in the presence of an electrode buffer containing 0.5% trichloroacetic acid.

"FIG. 4 shows fluorescence emitted by proteins in Gel D of the Example. No halo-substituted organic compound was incorporated into the gel upon pouring or present in the electrode buffer during electrophoresis."

For more information, see this patent application: Belisle, Christopher. Modified Electrode Buffers for Stain-Free Protein Detection in Electrophoresis. Filed August 7, 2013 and posted February 20, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=5585&p=112&f=G&l=50&d=PG01&S1=20140213.PD.&OS=PD/20140213&RS=PD/20140213

Keywords for this news article include: Peptides, Proteins, Treatment, Tryptophan, Legal Issues, Aromatic Amino Acids, Essential Amino Acids, Bio-Rad Laboratories Inc..

Our reports deliver fact-based news of research and discoveries from around the world. Copyright 2014, NewsRx LLC


For more stories covering the world of technology, please see HispanicBusiness' Tech Channel



Source: Life Science Weekly


Story Tools