Novel dyes and compositions, and processes for using same in analysis of protein aggregation and other applications

• US 20110130305A1
• Filed: 11/30/2009
• Published: 06/02/2011
• Est. Priority Date: 11/30/2009
• Status: Active Grant

• Current Assignee: Enzo Biochem Incorporated
• Original Assignee: Enzo Biochem Incorporated
• Inventors: Kovalska, Vladyslava, Volkova, Kateryna, Losytskyy, Mykhaylo, Yarmoluk, Sergiy M., Ludlum, Anthony, Coleman, Jack, Pande, Praveen, Balanda, Anatoliy, Dai, Lijun, Patton, Wayne Forrest
• Granted Patent: US 9,133,343 B2
• Time in Patent Office: Days
• US Class Current: 506/15
• CPC Class Codes: C07D 401/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C09B 23/0008 substituted on the polymethine chain
• CPC Class Codes: C09B 23/0025 the substituent being bound through an oxygen atom
• CPC Class Codes: C09B 23/0058 the substituent being CN
• CPC Class Codes: C09B 23/0066 the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
• CPC Class Codes: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/102 two heterocyclic rings linked carbon-to-carbon C09B7/00 takes precedence
• CPC Class Codes: C09B 23/141 Bis styryl dyes containing two radicals C6H5-CH=CH-
• CPC Class Codes: C09B 23/145 the ethylene chain carrying an heterocyclic residue, e.g. heterocycle-CH=CH-C6H5
• CPC Class Codes: G01N 2458/30 Electrochemically active labels
• CPC Class Codes: G01N 2800/2821 Alzheimer
• CPC Class Codes: G01N 2800/2835 Movement disorders, e.g. Parkinson, Huntington, Tourette
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

Provided are dyes and compositions which are useful in a number of applications, such as the detection and monitoring protein aggregation, kinetic studies of protein aggregation, neurofibrillary plaques analysis, evaluation of protein formulation stability, protein thermal stability shift assay and analysis of molecular chaperone activity. These dyes and compositions are also useful as probes in nucleic acid and protein detection.

Claims

1. A compound comprising S25, S43, TOL3, YAT2134, YAT2148, YAT2149, S13, YAT2135 or YAT2324.

2. A kit for assaying aggregation of a protein, comprising in packaged combination:

(a) one or more compounds from FIG. 1B;

(b) instructions therefor.

3. The kit of claim 2, further comprising:

(c) buffers;

or

(d) positive controls;

or

(e) negative controls, or

(f) a combination of any of the foregoing.

4. The kit of claim 3, further comprising additional instructions therefor.

5. The kit of claim 3, wherein said positive controls comprise protein aggregates and said negative controls comprise protein monomers.

6. The kit of claim 3, further comprising a microplate or a microarray.

7. A composition comprising any of the compounds from FIG. 1B or FIG. 2B, wherein said compound or compounds have been modified by the addition of a reactive group (Rx) for attachment of a target molecule thereto.

8. The composition of claim 7, wherein said reactive group (Rx) comprises an electrophilic reactive group comprising isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, 4,6,-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-propionamide, glyoxal or aldehyde groups, and a combination of any of the foregoing.

9. The composition of claim 7, wherein said reactive group (Rx) comprises a nucleophilic reactive group comprising reactive thiol, amine or hydroxyl, and a combination of the foregoing.

10. The composition of claim 7, wherein said reactive group (Rx) comprises a terminal alkene group, a terminal alkyne group, a nickel coordinate group or a platinum coordinate group for said attachment.

11. The composition of claim 7, wherein said reactive group (Rx) is attached to said compound through a linker arm.

12. A labeled target molecule comprising:

(a) a target molecule attached to

(b) any of the compounds from FIG. 1B or FIG. 2B, wherein said compound or compounds were modified by the addition of a reactive group (Rx) for attachment of said target molecule thereto.

13. The labeled target molecule of claim 12, wherein said target molecule comprises nucleoside, nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, streptavidin, digoxigenin, carbohydrate, oligosaccharide, polysaccharide, lipid, liposomes, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eukaryotic cell, eukaryotic cell component, natural drug or synthetic drug, and combinations of any of the foregoing.

14. The labeled target molecule of claim 12, wherein the reactive group for said attachment comprised an electrophilic reactive group comprising isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, 4,6,-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-propionamide, glyoxal or aldehyde groups, a nucleophilic reactive group comprising reactive thiol, amine or hydroxyl, a nickel coordinate group, a platinum coordinate group, a terminal alkene or a terminal alkyne, and any combination of the foregoing.

15. The labeled target molecule of claim 12, wherein a linker arm is between said compound and said reactive group.

16. The labeled target molecule of claim 12, wherein a linker arm is between said target molecule and said reactive group.

17. A composition comprising a solid support to which is attached any of the compounds from FIG. 1B or FIG. 2B, wherein said compound or compounds were modified by the addition of a reactive group (Rx) for attachment of said target molecule thereto.

18. The composition of claim 17, wherein said solid support comprises glass particle, glass surface, natural polymers, synthetic polymers, plastic particle, plastic surface, silicaceous particle, silicaceous surface, glass, plastic or latex beads, controlled pore glass, metal particle, metal oxide particle, microplate or microarray, and combinations of any of the foregoing.

19. The composition of claim 17, wherein the reactive group for said attachment comprised an electrophilic reactive group comprising isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, 4,6,-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-propionamide, glyoxal or aldehyde groups, a nucleophilic reactive group comprising reactive thiol, amine or hydroxyl, a nickel coordinate group, a platinum coordinate group, a terminal alkene or a terminal alkyne, and any combination of the foregoing.

20. The composition of claim 17, wherein a linker arm is between said compound and said reactive group.

21. The labeled target molecule of claim 17, wherein a linker arm is between said solid support and said reactive group.

22. A kit for assaying aggregation of a protein, comprising in packaged combination:

(a) two or more compounds, wherein one compound is from FIG. 1B and the other compound provides a higher intensity of fluorescence when measured in the presence of a protein aggregate as compared to the intensity of fluorescence when measured in the presence of a native monomeric form of said protein; and

(b) instructions therefor.

23. The kit of claim 22, further comprising:

(c) buffers;

or

(d) positive controls;

or

(e) negative controls, or

(f) a combination of any of the foregoing.

24. The kit of claim 23, wherein said positive controls comprise protein aggregates and said negative controls comprise protein monomers.

25. The kit of claim 22, wherein the emission maxima of said compounds is from about 600 nanometers to about 670 nanometers.

26. The kit of claim 22, wherein the emission maxima of said compounds differ by no more than about 50 nanometers (nm).

27. The kit of claim 26, wherein the emission maxima of said compounds differ by no more than about 10 nanometers (nm).

28. A kit for assaying aggregation of a protein, comprising in packaged combination:

(a) two or more compounds, wherein each of said compounds provides a higher intensity of fluorescence when measured in the presence of a protein aggregate as compared to the intensity of fluorescence when measured in the presence of a native monomeric form of said protein, and wherein the emission maxima of said compounds is within 50 nanometers (nm) of each other when measured in the presence of a protein aggregate; and

(b) instructions therefor.

29. The kit of claim 28, further comprising:

(c) buffers;

or

(d) positive controls;

or

(e) negative controls, or

(f) a combination of any of the foregoing.

30. The kit of claim 29, wherein said positive controls comprise protein aggregates and said negative controls comprise protein monomers.

31. The kit of claim 28, wherein the emission maxima of said compounds is from about 600 nanometers to about 670 nanometers.

32. The kit of claim 28, wherein the emission maxima of said compounds differ by no more than about 10 nanometers (nm).

33. The kit of claim 28, wherein at least one of said compounds comprises Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324.

34. A multi-dye composition comprising at least three dyes, wherein each of said at least three dyes in the presence of an aggregate of said protein has a higher florescent intensity as compared to the fluorescent intensity when measured in the presence of the native monomeric form of said protein.

35. A compound comprising any of D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324.

36. A multi-dye composition comprising two or more dyes, wherein at least one of said two or more dyes comprises Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324.

37. A method for detecting the presence of aggregates of a protein in a sample:

(i) providing;

(a) a sample;

(b) one or more dye compounds, wherein at least one of said dye compounds comprises Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324; and

(c) means for detecting fluorescence;

(ii) forming a mixture comprising said sample (a) and said one or more dye compounds (b); and

(iii) measuring the amount of fluorescence in said mixture, thereby detecting the presence of any protein aggregates in said sample.

38. The method of claim 37, wherein said sample (a) comprises tissue or cells or proteins derived therefrom, and combinations thereof.

39. The method of claim 37, wherein the amount of fluorescence measured in step (ii) is compared to the amount of fluorescence when measured in the absence of said sample (a).

40. The method of claim 37, wherein the amount of fluorescence measured in step (ii) is compared to the amount of fluorescence from a standard curve for protein aggregates and protein monomers in selected proportions.

41. The method of claim 40, wherein the protein for said standard curve is the same protein as the protein in said sample.

42. The method of claim 40, wherein the protein for said standard curve comprises a protein that is different from the protein in said sample.

43. A method for detecting the presence of aggregates of a protein in a sample:

(i) providing;

(a) a sample;

(b) one or more compositions having the formula

44. The method of claim 43, wherein said sample (a) comprises tissue or cells or proteins derived therefrom, and combinations thereof.

45. The method of claim 43, wherein the amount of fluorescence measured in step (ii) is compared to the amount of fluorescence when measured in the absence of said sample (a).

46. The method of claim 43, wherein the amount of fluorescence measured in step (ii) is compared to the amount of fluorescence from a standard curve for protein aggregates and protein monomers in selected proportions.

47. The method of claim 46, wherein the protein for said standard curve is the same protein as the protein in said sample.

48. The method of claim 46, wherein the protein for said standard curve comprises a protein that is different from the protein in said sample.

49. A method for detecting the formation of aggregates of a protein in a sample, said method comprising the steps of:

(i) providing;

(a) a sample;

(b) one or more of dye compounds, wherein at least one of said dye compounds comprises Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324; and

(c) means for detecting fluorescence;

(ii) forming a mixture with said sample (a) and said one or more dye compounds (b);

(iii) measuring at preselected time intervals the amount of fluorescence in said mixture formed in step (ii), thereby detecting the formation of protein aggregates.

50. The method of claim 49, wherein said sample (a) comprises tissue or cells or proteins derived therefrom, and combinations thereof.

51. The method of claim 49, wherein the amount of fluorescence measured in step (ii) is compared to the amount of fluorescence when measured in the absence of said sample (a).

52. The method of claim 49, wherein the amount of fluorescence measured in step (ii) is compared to the amount of fluorescence from a standard curve for protein aggregates and protein monomers in selected proportions.

53. The method of claim 52, wherein the protein for said standard curve is the same protein as the protein in said sample.

54. The method of claim 52, wherein the protein for said standard curve comprises a protein that is different from the protein in said sample.

55. The method of claim 49, wherein the prescribed intervals in said step (iii) and the prescribed intervals in said step (v) comprise minute intervals over the course of an hour.

56. A method for detecting the formation of aggregates of a protein in a sample, said method comprising the steps of:

(i) providing;

(a) a sample;

(b) one or more compositions having the formula

57. The method of claim 56, wherein said sample (a) comprises tissue or cells or proteins derived therefrom, and combinations thereof.

58. The method of claim 56, wherein the amount of fluorescence measured in step (ii) is compared to the amount of fluorescence when measured in the absence of said sample (a).

59. The method of claim 56, wherein the amount of fluorescence measured in step (ii) is compared to the amount of fluorescence from a standard curve for protein aggregates and protein monomers in selected proportions.

60. The method of claim 59, wherein the protein for said standard curve is the same protein as the protein in said sample.

61. The method of claim 59, wherein the protein for said standard curve comprises a protein that is different from the protein in said sample.

62. The method of claim 56, wherein the prescribed intervals in said step (iii) and the prescribed intervals in said step (v) comprise minute intervals over the course of an hour.

63. The method of claim 56, wherein the aggregates of said protein comprise aggresomes, aggresome-like structures, inclusion bodies, Lewy bodies, Mallory bodies or neurofibriliary tangles, and a combination of the foregoing.

64. A method for determining whether a test compound decreases aggregation of a protein, said method comprising the steps of:

(i) providing;

(a) said protein;

(b) one or more of compounds comprising Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324; and

(c) said test compound;

(ii) forming a first mixture comprising said protein (a) and said one or more compounds (b);

(iii) measuring the amount of fluorescence in said first mixture at prescribed intervals;

(iv) forming a second mixture comprising said protein (a), said one or more compounds (b) and said test compound (c);

(v) measuring the amount of fluorescence in said second mixture at prescribed intervals; and

(vi) comparing the amount of fluorescence measured in step (iii) and step (v);

thereby determining whether said test compound (c) decreases the aggregation of said protein (a).

65. The method of claim 64, wherein the prescribed intervals in said step (iii) and the prescribed intervals in said step (v) are the same intervals of time.

66. The method of claim 64, wherein the prescribed intervals are measured in minutes, hours or days.

67. The method of claim 64, wherein the prescribed intervals in said step (iii) and the prescribed intervals in said step (v) comprise minute intervals over the course of an hour.

68. The method of claim 64, wherein the prescribed intervals in said step (iii) and the prescribed intervals in said step (v) comprise daily intervals over the course of at least one month.

69. The method of claim 68, wherein step (iii), fluorescence is initially measured 30 minutes after forming the first mixture, and wherein step (v), fluorescence is initially measured 30 minutes after forming the second mixture.

70. The method of claim 67, wherein step (iii), fluorescence is measured in one or more 30 minute intervals after said initial measurement, and wherein step (v), fluorescence is measured in one or more 30 minute intervals after said initial measurement.

71. The method of claim 64, wherein after said forming steps (ii) and (iv), said first mixture and said second mixture are maintained at room temperature prior to measuring fluorescence in steps (iii) and (v).

72. The method of claim 64, wherein after said forming steps (ii) and (iv), said first mixture and said second mixture are incubated at a temperature ranging from about 4°

C. to about 95°

C.

73. The method of claim 72, wherein said first mixture and said second mixture are incubated at a temperature of about 30°

C. after said first mixture and said second mixture have been formed.

74. The method of claim 72, wherein said first mixture and said second mixture are incubated at a temperature of about 37°

C. after said first mixture and said second mixture have been formed.

75. The method of claim 64, wherein said test compound (c) comprises a kosmotrope, a chaotrope, an amino acid, a peptide, a reducing agent, a carbohydrate, a detergent, a surfactant, a zwitterion or a polyhydric alcohol, and combinations thereof.

76. The method of claim 75, wherein any of said test compounds (c) have a range of concentrations from about 0 molar to about 2 molar, a range of pH values from about 4 to about 10, and any combinations thereof.

77. The method of claim 64, wherein said test compound (c) comprises a storage buffer for said protein.

78. The method of claim 77, wherein said storage buffer comprises a set of buffer formulations with a range of concentrations from about 0 molar to about 2 molar, a range of pH values from about 4 to about 10, and any combinations thereof.

79. The method of claim 64, wherein said steps (iii) and (v), fluorescence is measured at one or more different temperatures after forming said first mixture and said second mixture.

80. The method of claim 79, wherein said different temperatures are selected from temperatures ranging from about 4°

C. to about 100°

C.

81. The method of claim 79, wherein said fluorescence measurements are carried out as a series of discrete temperatures, wherein said measuring steps (iii) and (v) are carried out after incubation at each of said different discrete temperatures.

82. The method of claim 79, wherein measuring steps (iii) and (v) are carried out while changing temperatures.

83. A method for determining whether a test compound decreases aggregation of a protein, said method comprising the steps of:

(i) providing;

(a) said protein;

(b) one or more compositions having the formula

84. The method of claim 83, wherein the prescribed intervals in said step (iii) and the prescribed intervals in said step (v) are the same intervals of time.

85. The method of claim 83, wherein the prescribed intervals are measured in minutes, hours or days.

86. The method of claim 83, wherein the prescribed intervals in said step (iii) and the prescribed intervals in said step (v) comprise minute intervals over the course of an hour.

87. The method of claim 83, wherein the prescribed intervals in said step (iii) and the prescribed intervals in said step (v) comprise daily intervals over the course of at least one month.

88. The method of claim 87, wherein step (iii), fluorescence is initially measured 30 minutes after forming the first mixture, and wherein step (v), fluorescence is initially measured 30 minutes after forming the second mixture.

89. The method of claim 86, wherein step (iii), fluorescence is measured in one or more 30 minute intervals after said initial measurement, and wherein step (v), fluorescence is measured in one or more 30 minute intervals after said initial measurement.

90. The method of claim 83, wherein after said forming steps (ii) and (iv), said first mixture and said second mixture are maintained at room temperature prior to measuring fluorescence in steps (iii) and (v).

91. The method of claim 83, wherein after said forming steps (ii) and (iv), said first mixture and said second mixture are incubated at a temperature ranging from about 4°

C. to about 95°

C.

92. The method of claim 91, wherein said first mixture and said second mixture are incubated at a temperature of about 30°

C. after said first mixture and said second mixture have been formed.

93. The method of claim 91, wherein said first mixture and said second mixture are incubated at a temperature of about 37°

C. after said first mixture and said second mixture have been formed.

94. The method of claim 83, wherein said test compound (c) comprises a kosmotrope, a chaotrope, an amino acid, a peptide, a reducing agent, a carbohydrate, a detergent, a surfactant, a zwitterion or a polyhydric alcohol, and combinations thereof.

95. The method of claim 94, wherein any of said test compounds (c) have a range of concentrations from about 0 molar to about 2 molar, a range of pH values from about 4 to about 10, and any combinations thereof.

96. The method of claim 83, wherein said test compound (c) comprises a storage buffer for said protein.

97. The method of claim 96, wherein said storage buffer comprises a set of buffer formulations with a range of concentrations from about 0 molar to about 2 molar, a range of pH values from about 4 to about 10, and any combinations thereof.

98. The method of claim 83, wherein said steps (iii) and (v), fluorescence is measured at one or more different temperatures after forming said first mixture and said second mixture.

99. The method of claim 98, wherein said different temperatures are selected from temperatures ranging from about 4°

C. to about 100°

C.

100. The method of claim 98, wherein said fluorescence measurements are carried out as a series of discrete temperatures, wherein said measuring steps (iii) and (v) are carried out after incubation at each of said different discrete temperatures.

101. The method of claim 98, wherein measuring steps (iii) and (v) are carried out while changing temperatures.

102. A method for determining whether a test compound affects aggregation of a protein, said method comprising the steps of:

(i) providing;

(a) said protein;

(b) two or more dyes wherein each of said dyes has a fluorescence intensity that is at least three times higher when measured in the presence of an aggregate of a protein as compared to the fluorescence intensity when measured in the presence of a native monomer of said protein;

(c) a test compound; and

(d) means for detection of fluorescence;

(ii) forming a first mixture with said protein (a) and said two or more dyes (b);

(iii) measuring the amount of fluorescence in said first mixture at prescribed intervals;

(iv) forming a second mixture with said protein (a), said two or more dyes (b) and said compound to be assayed (c);

(v) measuring the amount of fluorescence in said second mixture at prescribed intervals; and

(vi) comparing the amount of fluorescence measured in step (iii) and step (v), thereby determining whether said test compound (c) affects aggregation of said protein.

103. The method of claim 102, wherein said dyes in the presence of a protein aggregate have emission maxima within 150 nm of each other.

104. The method of claim 103, wherein said emission maxima is within 50.

105. The method of claim 102, wherein at least one of said two or more dyes comprises a compound having a structure from FIG. 1B.

106. A method for determining whether a test compound affects aggregation of a protein, said method comprising the steps of:

(i) providing;

(a) said protein;

(b) two or more dyes, wherein each of said two or more dyes in the presence of an aggregate of said protein has a higher florescent intensity as compared to the fluorescent intensity when measured in the presence of the native monomeric form of said protein, and wherein at least one of said dyes has the formula

107. The method of claim 106, wherein said at least one dye having the formula, further has a structure from FIG. 1B.

108. A method for determining whether a test compound affects aggregation of a protein, said method comprising the steps of:

(i) providing;

(a) said protein;

(b) two or more dyes, wherein at least one of said dyes comprises Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324, and wherein each of said two or more dyes in the presence of an aggregate of said protein has a higher florescent intensity as compared to the fluorescent intensity when measured in the presence of the native monomeric form of said protein;

(c) a compound to be assayed; and

(d) means for detecting fluorescence;

(ii) forming a first mixture with said protein (a) and said two or more dyes (b);

(iii) measuring the amount of fluorescence in said first mixture at prescribed intervals;

(iv) forming a second mixture with said protein (a), said two or more dyes (b) and said compound to be assayed (c);

(v) measuring the amount of fluorescence in said second mixture at prescribed intervals; and

(vi) comparing the amount of fluorescence measured in step (iii) and step (v), thereby determining whether said test compound (c) affects aggregation of said protein.

109. The method of claim 108, wherein said dyes in the presence of a protein aggregate have emission maxima within 150 nm of each other.

110. The method of claim 109, wherein said emission maxima is within 50 nm.

111. A method of determining temperature dependency of aggregation of a protein, said method comprising the steps of:

(i) providing;

(a) said protein;

(b) two or more dyes, wherein each of said two or more dyes in the presence of an aggregate of said protein has a higher florescent intensity as compared to the fluorescent intensity when measured in the presence of the native monomeric form of said protein, and wherein at least one of said two or more dyes is selected from S13, S25, S39, S42, S43, TOL-2, TOL-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 and YAT2324;

(c) means for detecting fluorescence;

(ii) forming a first mixture with said protein (a) and said two or more dyes (b);

(iii) measuring the amount of fluorescence in said first mixture at prescribed intervals;

(iv) heating said first mixture and measuring the amount of fluorescence continuously or incrementally as the temperature of said first mixture is raised; and

(iv) comparing the measurements of fluorescence as the temperature is raised in step (iv) with the amount of fluorescence measured in step (iii), thereby determining the temperature dependency of aggregation of said protein.

112. The method of claim 111, further comprising a test compound (d) for determining whether said test compound decreases aggregation.

113. The method of claim 111, wherein step (iv), heating is carried out in a temperature range of from about 4°

C. to about 95°

C.

114. The method of claim 111, wherein step (iv), incremental measuring is carried out as the temperature is raised in increments of 1°

C., 5°

C. or 10°

C.

115. A method of determining temperature dependency of aggregation of a protein, said method comprising the steps of:

(i) providing;

(a) said protein;

(b) two or more dyes, wherein each of said two or more dyes in the presence of an aggregate of said protein has a higher florescent intensity as compared to the fluorescent intensity when measured in the presence of the native monomeric form of said protein, and wherein at least one of said dyes has the formula

116. The method of claim 115, wherein said dyes in the presence of a protein aggregate have emission maxima within 150 nm of each other.

117. The method of claim 116, wherein said emission maxima is within 50 nm.

118. The method of claim 115, wherein said at least one dye having the formula, further has a structure from FIG. 1B.

119. A method of determining temperature dependency of aggregation of a protein, said method comprising the steps of:

(i) providing;

(a) said protein;

(b) two or more dyes, wherein each of said two or more dyes in the presence of an aggregate of said protein has a higher florescent intensity as compared to the fluorescent intensity when measured in the presence of the native monomeric form of said protein, and wherein said dyes have emission maxima within 150 nm of each other in the presence of an aggregate of said protein; and

(c) means for detecting fluorescence;

(ii) forming a first mixture with said protein (a) and said two or more dyes (b);

(iii) measuring the amount of fluorescence in said first mixture;

(iv) heating said first mixture and measuring the amount of fluorescence continuously or incrementally as the temperature is raised; and

(iv) comparing the measurements of fluorescence in step (iv) with the amount of fluorescence measured in step (iii), thereby determining the temperature dependency of aggregation of said protein.

120. The method of claim 119, wherein at least one of said two or more dyes comprises a compound having a structure from FIG. 1B.

121. The method of claim 119, wherein said emission maxima is within 50 nm.

122. A method for measuring chaperone-like activity, the method comprising the steps of:

(i) forming a reaction mixture comprising;

(a) a chaperone;

(b) a substrate for said chaperone;

(c) one or more of compounds comprising Dye F(?), Dye Fm(b)(?), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324;

(ii) exposing said reaction mixture to a stress for a period of time sufficient to induce aggregation of said substrate (b); and

(iii) measuring the fluorescence intensity of said exposed mixture, wherein a decrease in said fluorescence intensity compared with the fluorescence intensity of a negative control is indicative of chaperone activity.

123. The method of claim 122, wherein said chaperone comprises a member selected from conserved classes and small heat-shock proteins (sHSPs).

124. The method of claim 123, wherein said conserved classes comprise HSP33, HSP60, HSP70, HSP90 or HSP100, and combinations thereof.

125. The method of claim 122, wherein said chaperone comprises GRP94, GRP170, calnexin, calreticulin, HSP 40, HSP47 and ERp29, GroEL, GroES, HSP60, Cpn10, DnaK, DnaJ, Hsp70, Hsp71, Hsp72, Grp78 (BiP), PDI, Erp72, Hsx70, Hdj1, Hdj2, Mortalin, Hsc70, Hsp70-A1, fHtpG, C62.5, Hsp90 alpha, Hsp90 beta, Grp94, ClpB, ClpA, ClpX, Hsp100, Hsp104, Hsp110, TRiC, alpha crystallin, HspB1, Hsp 25, Hsp27, clusterin, GrpE, Trigger Factor, or Survival of Motor Neuron (SMN1, SMN2), and combinations thereof.

126. The method of claim 122, wherein said substrate (b) comprises is R-lactoglobulin, citrate synthase, lysozyme, immunoglobulin, CRYBB2, HSPB8, CRYAA, TGFB1I1, HNRPD or CRYAB, and combinations thereof.

127. The method of claim 122, wherein said reaction mixture is incubated for a period of time from about 15 to about 60 minutes.

128. The method of claim 122, wherein said stress is an elevated temperature.

129. The method of claim 128, wherein said elevated temperature is from about 37°

C. to about 95°

C.

130. The method of claim 122, wherein said stress is a chaotropic agent.

131. The method of claim 130, wherein said chaotropic agent comprises guanidine-HCl or urea, or both.

132. The method of claim 130, wherein the concentration of said chaotropic agent is from about 4 to 8 M.

133. The method of claim 122, wherein a plurality of said methods are performed in parallel.

134. A method for measuring activity of a member of the thioredoxin superfamily, the method comprising the steps of:

(i) forming a reaction mixture comprising;

(a) a member of the thioredoxin superfamily;

(b) a substrate for said member of the thioredoxin superfamily;

(c) a reducing agent; and

(d) one or more of compounds comprising Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324;

(ii) incubating said reaction mixture for a period of time sufficient to reduce disulfide bonds in said substrate; and

(iii) measuring the fluorescence intensity of said mixture, wherein an increase in said fluorescence intensity compared with the fluorescence intensity of a negative control is indicative of activity of said member of the thioredoxin superfamily.

135. The method of claim 134, wherein said member of the thioredoxin superfamily (a) comprises a protein disulfide isomerase, a thioredoxin or a glutaredoxin, and combinations thereof.

136. The method of claim 134, wherein said substrate (b) comprises insulin ribonuclease, choriogonadotropin, coagulation factor, glucocorticoid receptor or HIV gp 120, and combinations thereof.

137. The method of claim 134, wherein said reducing agent (c) is selected from the group dithiothreitol (DTT), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP HCl) or dithioerythritol (DTE), and combinations thereof.

138. The method of claim 134, wherein said reaction mixture is incubated for a period of time from about 15 to about 60 minutes.

139. The method of claim 135, wherein said protein disulfide isomerase comprises PDI, ERp57, PDIp, ERp72, P5, PDIr, ERp28/29, ERp44, ERjd5/JPDI or ERp18, and combinations thereof.

140. The method of claim 134, further comprising the step of terminating the reaction prior to said measuring step (iii) by adding hydrogen peroxide to the incubating reaction mixture.

141. The method of claim 134, wherein a plurality of said methods are performed in parallel.

142. A method for separating aggregates of proteins from monomeric forms of said proteins, said method comprising the steps of:

(i) providing;

(a) a sample that having aggregates of said proteins and monomeric forms of said proteins;

(b) one or more of compounds, wherein at least one of said compounds is selected from Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 and YAT2324, and wherein said one or more compounds are attached to a solid matrix;

(ii) forming under binding conditions a mixture with said sample (a) and said one or more dyes (b) to allow binding between said one or more compounds (b) and any aggregates of said proteins in said sample (a); and

(iii) separating unbound proteins from the aggregates bound to said one or more compounds (a) in step (ii).

143. The composition of claim 142, wherein said solid support comprises glass particle, glass surface, natural polymers, synthetic polymers, plastic particle, plastic surface, silicaceous particle, silicaceous surface, glass, plastic or latex beads, controlled pore glass, metal particle, metal oxide particle, microplate or microarray, and combinations of any of the foregoing.

144. A method for separating aggregates of proteins from monomeric forms of said proteins, said method comprising the steps of:

(i) providing;

(a) a sample that having aggregates of said proteins and monomeric forms of said proteins;

(b) two or more dyes, wherein each of said two or more dyes in the presence of an aggregate of said protein has a higher florescent intensity as compared to the fluorescent intensity when measured in the presence of the native monomeric form of said protein, and wherein at least one of said dyes has the formula

145. The composition of claim 144, wherein said solid support comprises glass particle, glass surface, natural polymers, synthetic polymers, plastic particle, plastic surface, silicaceous particle, silicaceous surface, glass, plastic or latex beads, controlled pore glass, metal particle, metal oxide particle, microplate or microarray, and combinations of any of the foregoing.