Compositions, methods and kits for biarsenical fluorophore labeling

Abstract

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 a protein sample with a biarsenical fluorophore, the method comprising: 
 treating the proteins in said sample in the presence of a composition comprising a thiol competitor and a biarsenical fluorophore.    
     
     
         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 1 , wherein said thiol competitor is a monothiol.  
     
     
         5 . The method of  claim 4 , wherein said monothiol is beta-mercaptoethanol (BME) and the BME is at a concentration greater than about 1 mM.  
     
     
         6 . The method of  claim 5 , wherein said BME is at a concentration of at least about 5 mM.  
     
     
         7 . The method of  claim 6 , wherein said BME is at a concentration of at least about 60 mM.  
     
     
         8 . The method of  claim 1 , wherein said thiol competitor is a dithiol with concentration of at least 100 μM.  
     
     
         9 . (canceled)  
     
     
         10 . The method of  claim 2 , wherein said reducing agent is at a concentration of at least about 1 mM.  
     
     
         11 - 12 . (canceled)  
     
     
         13 . The method of  claim 1 , wherein said biarsenical fluorophore is a biarsenical derivative of fluorescein.  
     
     
         14 . The method of  claim 13 , wherein said biarsenical fluorophore is 4′-5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(2,2-ethanedithiol)2.  
     
     
         15 . (canceled)  
     
     
         16 . The method of  claim 2 , wherein said biarsenical fluorophore is added to said protein sample after said thiol competitor and said reducing agent are added.  
     
     
         17 - 19 . (canceled)  
     
     
         20 . The method of  claim 16 , wherein said treating step is at a temperature of about 70° C.  
     
     
         21 - 24 . (canceled)  
     
     
         25 . The method of  claim 1 , wherein said treating is for a period of no more than about 10 minutes.  
     
     
         26 . The method of  claim 1 , further comprising a later step of: 
 resolving the proteins in said sample by gel electrophoresis.    
     
     
         27 . The method of  claim 26 , comprising a yet further step of detecting said tetracysteine-tagged protein among said resolved proteins in said gel.  
     
     
         28 . The method of  claim 1 , further comprising a later step of: 
 treating said protein sample with a thiol-blocking reagent.    
     
     
         29 . The method of  claim 28 , 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.  
     
     
         30 . (canceled)  
     
     
         31 . The method of  claim 29 , wherein said thiol-blocking reagent is maleimide.  
     
     
         32 - 39 . (canceled)  
     
     
         40 . The method of  claim 1 , wherein said biarsenical fluorophore is bonded to a surface of a support.  
     
     
         41 . The method of  claim 40 , wherein said support comprises a plurality of beads.  
     
     
         42 - 47 . (canceled)  
     
     
         48 . A composition for improving specificity of labeling of tetracysteine-tagged proteins with a biarsenical fluorophore, comprising: 
 a biarsenical fluorophore; and    a thiol competitor.    
     
     
         49 . The composition of  claim 48  further comprising a reducing agent, wherein said reducing agent is tributylphosphine (TBP).  
     
     
         50 . The composition of  claim 48 , wherein said thiol competitor is a mono- or dithiol.  
     
     
         51 . The composition of  claim 50 , wherein said thiol competitor is beta-mercaptoethanol (BME).  
     
     
         52 . A kit for labeling tetracysteine-tagged proteins with a biarsenical fluorophore, comprising: 
 a first composition comprising a thiol competitor.    
     
     
         53 . The kit of  claim 52 , wherein said first composition further comprises a biarsenical fluorophore.  
     
     
         54 . The kit of  claim 52 , further comprising a second composition comprising a biarsenical fluorophore.  
     
     
         55 . The kit of  claim 52 , further comprising: 
 a composition comprising a blocking reagent.    
     
     
         56 . The kit of  claim 52 , wherein said thiol competitor is a mono- or dithiol.  
     
     
         57 . The kit of  claim 56 , wherein said thiol competitor is beta-mercaptoethanol (BME).  
     
     
         58 . The kit of  claim 52 , wherein said first composition further comprises a reducing agent, wherein said reducing agent is a phosphine.  
     
     
         59 . The kit of  claim 58 , wherein said phosphine is tributylphosphine (TBP).  
     
     
         60 . The kit of  claim 52 , wherein said biarsenical fluorophore is a biarsenical fluorescein derivative.  
     
     
         61 . (canceled)  
     
     
         62 . The kit of  claim 55 , wherein said blocking reagent is selected from maleimide and iodoacetamide.  
     
     
         63 . The kit of  claim 62 , where said blocking reagent is maleimide.  
     
     
         64 - 67 . (canceled)  
     
     
         68 . The composition of  claim 48  further comprising at least one buffer, an ionic detergent, and a marker dye.  
     
     
         69 . The composition of  claim 68 , wherein the buffer comprises Tris and EDTA; and the ionic detergent is a dodecylsulfate salt.