Compositions, methods and kits for biarsenical fluorophore labeling
View Patent ↗Methods, compositions, and kits for labeling tetracysteine-tagged proteins with biarsenical fluorophores with increased specificity, including compositions, methods and kits particularly adapted for labeling of tetracysteine-tagged proteins to be resolved within an electrophoresis gel.
1. A method of labeling a tetracysteine-tagged protein species present in an inhomogeneous protein sample with a biarsenical fluorophore, the method comprising:
a) treating the proteins in said sample in the presence of a composition comprising a thiol competitor and a biarsenical fluorophore, wherein each arsenic of the biarsenical fluorophore is bonded to a dithiol prior to said treating; and
b) resolving the proteins in said sample by electrophoresis.
2. The method of claim 1 , wherein said composition further comprises a reducing agent.
3. The method of claim 2 , wherein said reducing agent is tributylphosphine (TBP).
4. The method of claim 2 , wherein said reducing agent is at a concentration of at least about 10 μM.
5. The method of claim 4 , wherein said reducing agent is at a concentration of at least about 1 mM.
6. The method of claim 5 , wherein said reducing agent is at a concentration of at least about 10 mM.
7. The method of claim 6 , wherein said reducing agent is at a concentration of at least about 100 mM.
8. The method of claim 2 , wherein said biarsenical fluorophore is added to said sample after said thiol competitor and said reducing agent are added.
9. The method of claim 2 , further comprising a step of:
treating said sample with a thiol-blocking reagent after said treating of a) and before said resolving of b).
10. The method of claim 9 , wherein said thiol-blocking reagent is selected from the group consisting of: alkylhalides, iodoacetamide, iodoacetic acid, dithiobis(2-nitrobenzoic acid) (DTNB), dithiobis(5-nitropyridine), maleimide and ethylenemaleimide.
11. The method of claim 10 , wherein said thiol-blocking reagent is iodoacetamide.
12. The method of claim 10 , wherein said thiol-blocking reagent is maleimide.
13. The method of claim 9 , wherein said thiol competitor is a mono- or di-thiol, and said thiol-blocking reagent is present at a concentration insufficient to block all of said thiol competitor thiols.
14. The method of claim 13 , wherein said thiol competitor is beta-mercaptoethanol (BME), said reducing agent is tributylphosphine (TBP), and said thiol-blocking agent is either iodoacetamide or maleimide.
15. The method of claim 9 , wherein said electrophoresis is in a gel having substantially neutral pH.
16. The method of claim 15 , wherein said gel is a Bis-Tris gel.
17. The method of claim 15 , comprising a yet further step of detecting said tetracysteine-tagged protein among said resolved proteins in said gel.
18. The method of claim 17 , wherein said detecting comprises exciting said fluorophore near its excitation maximum and observing fluorescence emission near its emission maximum.
19. The method of claim 18 , wherein said gel is free of mechanical supports within which the gel was cast and within which the gel was electrophoresed.
20. The method of claim 1 , wherein said thiol competitor is a monothiol.
21. The method of claim 20 , wherein said monothiol is beta-mercaptoethanol (BME) and the BME is at a concentration greater than about 1 mM.
22. The method of claim 21 , wherein said BME is at a concentration of at least about 5 mM.
23. The method of claim 22 , wherein said BME is at a concentration of at least about 60 mM.
24. The method of claim 1 , wherein said thiol competitor is a dithiol with concentration of at least 100 μM.
25. The method of claim 1 , wherein said biarsenical fluorophore is a biarsenical derivative of fluorescein.
26. The method of claim 25 , wherein said biarsenical fluorophore is 4′-5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(2,2-ethanedithiol)2.
27. The method of claim 1 , wherein said biarsenical fluorophore is a derivative of resorufin.
28. The method of claim 1 , wherein said treating step is performed at temperatures above room temperature.
29. The method of claim 28 , wherein said treating step is at a temperature of at least about 42° C.
30. The method of claim 29 , wherein said treating step is at a temperature of at least about 50° C.
31. The method of claim 30 , wherein said treating step is at a temperature of about 70° C.
32. The method of claim 1 , wherein said treating of said proteins in the presence of a thiol competitor and biarsenical fluorophore is performed for a period of no more than about 2 hours.
33. The method of claim 32 , wherein said treating is performed for a period of no more than about 1 hour.
34. The method of claim 33 , wherein said treating is performed for a period of no more than about 30 minutes.
35. The method of claim 34 , wherein said treating is for a period of no more than about 15 minutes.
36. The method of claim 35 , wherein said treating is for a period of no more than about 10 minutes.
37. The method of claim 1 , wherein,
said resolving is by gel electrophoresis.
38. The method of claim 37 , comprising a yet further step of detecting said tetracysteine-tagged protein among said resolved proteins in said gel.
39. The method of claim 38 , wherein said detecting comprises exciting said fluorophore near its excitation maximum and observing fluorescence emission near its emission maximum.
40. The method of claim 39 , wherein said gel is free of mechanical supports within which the gel was cast and within which the gel was electrophoresed.
41. The method of claim 1 , wherein said sample is a cell lysate.