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  Use of dye to distinguish salt and protein crystals under microcrystallization conditionsUSPTO Application #: 20070111317Title: Use of dye to distinguish salt and protein crystals under microcrystallization conditions Abstract: An improved method of screening crystal growth conditions is provided wherein molecules are crystallized from solutions containing dyes. These dyes are selectively incorporated or associated with crystals of particular character thereby rendering crystals of particular character colored and improving detection of the dyed crystals. A preferred method involves use of dyes in protein solutions overlayed by oil. Use of oil allows the use of small volumes of solution and facilitates the screening of large numbers of crystallization conditions in arrays using automated devices that dispense appropriate solutions to generate crystallization trials, overlay crystallization trials with an oil, provide appropriate conditions conducive to crystallization and enhance detection of dyed (colored) or undyed (uncolored) crystals that result. (end of abstract) Agent: Bingham, Mccutchen LLP - Washington, DC, US Inventors: Larry Cosenza, Terry L. Bray, Lawrence J. Delucas, Thomas E. Gester, David T. Hamrick USPTO Applicaton #: 20070111317 - Class: 436086000 (USPTO) Related Patent Categories: Chemistry: Analytical And Immunological Testing, Peptide, Protein Or Amino Acid The Patent Description & Claims data below is from USPTO Patent Application 20070111317. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority of U.S. Provisional Application No. 60/308,698, filed Jul. 30, 2001, to Provisional Application Ser. No. 60/328,958, filed Oct. 12, 2001, and to U.S. application Ser. No. 09/543,326, filed Apr. 5, 2000, which claims benefit of U.S. Provisional Application 60/128,018, filed Apr. 6, 1999, each of which are hereby incorporated in their entirety herein by reference. FIELD OF THE INVENTION [0003] This invention relates generally to the method of using detectable agents in crystallization of proteins, wherein the detectable agents are preferentially incorporated in the crystals of proteins or preferentially not incorporated in the crystals of proteins such that protein crystals can be readily distinguished from crystals of other substances which can form under the crystallization conditions, thereby allowing rapid and straightforward characterization of crystals and evaluation of crystallization conditions used to obtain the crystals. SUMMARY OF THE INVENTION [0004] In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention relates to a method of using dyes to facilitate the characterization of protein crystals present in volumes of one microliter or less. Specifically, it relates to the crystallization of proteins from protein solutions containing dyes under conditions wherein the proteins which crystallize and the other components of the protein solutions which crystallize can be distinguished from one another on the basis of whether or not they form dyed crystals. [0005] In a first aspect, this invention relates to a method for forming a dyed protein crystal including the steps of: (1) providing a protein solution, wherein the protein solution contains at least one dye, which dye is capable of dyeing at least a portion of protein crystals formed from the protein solution; (2) subjecting the protein solution to environmental conditions effective to from protein crystals; and (3) detecting the presence of dyed protein crystals, whereby the presence of the dyed protein crystals indicates the presence of protein crystals. [0006] In various preferred embodiments of the first aspect of the invention, the dye can be chosen from the group consisting of methylene blue, methylene green, Izit.sup.1 and crystal violet. .sup.1Reagent is commercially available from Hampton Research, Inc. (www.hamptonresearch.com) [0007] In various preferred embodiments of the first aspect of the invention, the protein solution is partitioned from the atmosphere. When the protein solution is partitioned from the atmosphere, the partitioning can be done so as to lower the rate at which transfer of solvent from the protein solution occurs. The protein solution can be partitioned from the atmosphere by overlaying the protein solution with an oil. The overlaying oil can be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL's oil.sup.1 (a 1:1 mixture of paraffin and silicone oil). The particular mixture of oil selected can be optimized for the rate at which transfer of solvent from a protein solution Occurs when the protein solution is overlayed with a particular mixture of oil. Further, the amount of oil overlaying the protein solution can be selected so as to optimize the rate of solvent transfer from the protein solution, in particular, from the protein solution to the oil and from the oil to the atmosphere. [0008] In various preferred embodiments of the first aspect of the invention, the protein solution further includes a component selected from the group consisting of salts, bluffers, precipitants, crystallization aids and any combination thereof. The component selected can crystallize under the controlled environmental conditions to which the protein solution is subjected, thereby producing component crystals. The conditions under which the component crystallizes, can include the step or process of adding a precipitate solution. The component crystals formed can be undyed component crystals. [0009] In various preferred embodiments of the first aspect of the invention, any crystals formed in the provided protein solution can be detected. If crystals are detected, dyed protein crystals can be distinguished from undyed component crystals. The protein crystals and component crystals can be detected by microscopy. [0010] In a second aspect, this invention relates to a method for screening protein crystal growth conditions, including the steps of: (1) providing a set of at least two protein solutions, wherein the protein solutions contain a dye, which is capable of dyeing at least a portion of protein crystals formed from the protein solution and which does not dye a significant portion of component crystals formed from the protein solution; (2) subjecting the protein solutions to predetermined conditions, wherein the environmental conditions to which each member of the set of protein solutions is subjected is not identical to the environmental conditions to which another member of the set is subjected; and (3) detecting the presence or non-presence of dyed protein crystals and/or undyed component crystals, wherein the presence of the dyed protein crystals indicates the formation of protein crystals; and the presence of undyed component crystals indicates formation of component crystals. [0011] In various preferred embodiments of the second aspect of the invention, the set of protein solutions consists of greater than 10, 50, 100, 250, 500, 1000, 1500, 2000 or 5000 protein solutions. [0012] In various preferred embodiments of the second aspect of the invention, the dye can be chosen from the group consisting of methylene blue, methylene green, Izit.sup.1 and crystal violet. [0013] In various preferred embodiments of the second aspect of the invention, the protein solutions are partitioned from the atmosphere. When the protein solutions are partitioned from the atmosphere, the partitioning can be done so as to lower the rate at which transfer of solvent from the protein solutions occurs. The protein solutions can be partitioned from the atmosphere by overlaying the protein solutions with an oil. The overlaying oil can be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL's oil.sup.1 (a 1:1 mixture of paraffin and silicone oil). The particular mixture of oil selected can be optimized for the rate at which transfer of solvent from a protein solution occurs when the protein solution is overlayed with a particular mixture of oil. Further, the amount of oil overlaying the protein solution can be selected so as to optimize the rate of solvent transfer from the protein solution, in particular, from the protein solution to the oil and from the oil to the atmosphere. [0014] In various preferred embodiments of the second aspect of the invention, the protein solutions further include a component selected from the group consisting of salts, buffers, precipitants, crystallization aids and any combination thereof. The component selected can crystallize under the controlled environmental conditions to which the protein solution is subjected, thereby producing component crystals. The conditions under which the component crystallizes, can include the step or process of adding a precipitate solution. The component crystals formed can be undyed component crystals. [0015] In various preferred embodiments of the second aspect of the invention, any crystals formed in the provided protein solutions can be detected. If crystals are detected, dyed protein crystals can be distinguished from undyed component crystals. The protein crystals and component crystals can be detected by microscopy. [0016] In various preferred embodiments of the second aspect of the invention, the detection of dyed protein crystals indicates a combination of environmental conditions to promote crystal growth of a protein in the provide protein solutions. When dyed proteins are detected, the dyed protein crystals can be evaluated in respect to protein crystal quality. Evaluation of protein crystal quality can include consideration of criteria selected from the group consisting of, but not limited to, size of crystals, volume of crystals, intensity of coloration of crystals by dye, color of coloration of crystals by dye, sharpness of crystal edges, and crystal shape. [0017] In various preferred embodiments of the second aspect of the invention, the suitability of crystallization conditions can include evaluation of the protein crystal quality of the crystals obtained by a certain set of environmental conditions. [0018] In a third aspect, this invention relates to a method of forming a dyed component crystal including the steps of: (1) providing a component solution, wherein the component solution contains at least one dye, which dye is capable of dyeing at least a portion of component crystals formed from the component solution; (2) subjecting the component solution to environmental conditions effective to form component crystals; and (3) detecting the presence of dyed component crystals, whereby the presence of the dyed component crystals indicates the presence of component crystals. [0019] In various preferred embodiments of the third aspect of the invention, the component solution is partitioned from the atmosphere. When the component solution is partitioned from the atmosphere, the partitioning can be done so as to lower the rate at which transfer of solvent to or from the component solution occurs. The component solution can be partitioned from the atmosphere by overlaying the component solution with an oil. The overlaying oil can be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL's oil.sup.1 (a 1:1 mixture of paraffin and silicone oil). The particular mixture of oil selected can be optimized for the rate at which transfer of solvent to or from the component solution occurs when the component solution is overlayed with a particular mixture of oil. Further, the amount of oil overlaying the component solution can be selected so as to optimize the rate of solvent transfer to or from the component solution, in particular, from the component solution to the oil, or vice versa, and from the oil to the atmosphere, or vice versa. [0020] In various preferred embodiments of the third aspect of the invention, the component solution further includes a protein. The protein selected can crystallize under the controlled environmental conditions to which the component solution is subjected, thereby producing protein crystals. The conditions under which the protein crystallizes, can include the step or process of adding a precipitate solution. The protein crystals formed can be dyed protein crystals. [0021] In various preferred embodiments of the third aspect of the invention, any crystals formed in the provided component solution can be detected. If crystals are detected, protein crystals can be distinguished from component crystals on the basis that the protein crystals are undyed protein crystals and the component crystals are dyed component crystals. The protein crystals and component crystals can be detected by microscopy. [0022] In a fourth aspect, this invention relates to a method for screening protein crystal growth conditions including the steps of: (1) providing a set of at least two protein solutions, wherein the protein solutions contain a dye, which is capable of dyeing at least a portion of component crystals formed from the protein Solution and which does not dye a significant portion of protein crystals formed from the protein solution; (2) subjecting the protein solutions to predetermined conditions, wherein the environmental conditions to which each member of the set of protein solutions is subjected is not identical to the environmental conditions to which another member of the set is subjected; and (3) detecting the presence or non-presence of undyed protein crystals and/or dyed component crystals. The presence of the undyed protein crystals can indicate the formation of protein crystals and the presence of the dyed component crystals can indicate formation of component crystals. 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