Fluorescent pI Markers for Isoelectric Focusing Separations and Fluorescent Labeling

Abstract

UV-Absorbing and fluorescent pI markers for isoelectric focusing separations and fluorescent labeling, and methods for making and using the markers.

1 - 16 . (canceled) 
     
     
         17 . A compound having the formula: 
       
         
           
           
               
               
           
         
         wherein 
         R is selected from the group consisting of 
         —(CH 2 ) n — where n is from 1 to 12, 
         —(CH 2 CH<) n — where n is from 1 to 12, 
         —(CH 2 CH 2 O) n — where n is from 1 to 20, and 
         —(CH 2 CH(OH)CH 2 O) n — where n is from 1 to 20, 
         wherein each is coupled to hydrogen, a non-charged group, or at least one moiety selected from the group consisting of amino, secondary amine, tertiary amine, quaternary amine, azaaryl, hydroxyaryl, carboxylic acid or carboxylate, sulfonic acid or sulfonate, and or hydrogen sulfate or sulfate group; and 
         R 1  and R 2  at each occurrence are independently selected from the group consisting of 
         —(CH 2 ) n - where n is from 1 to 12, 
         —(CH 2 CH<) n - where n is from 1 to 12, 
         —(CH 2 CH 2 O) n — where n is from 1 to 20, and 
         —(CH 2 CH(OH)CH 2 O) n - where n is from 1 to 20, 
         wherein each is coupled to hydrogen, a non-charged group, or at least one moiety selected from the group consisting of amino, secondary amine, tertiary amine, azaaryl, hydroxyaryl, and carboxylic acid group. 
       
     
     
         18 - 21 . (canceled) 
     
     
         22 . The compound of  claim 17 , wherein Ri at each occurrence is the same. 
     
     
         23 . The compound of  claim 17 , wherein Ri at each occurrence is different. 
     
     
         24 . The compound of  claim 17 , wherein Ri at each occurrence comprises a group independently selected from the group consisting of amino, secondary amino, tertiary amino, azaryl, hydroxyaryl, carboxylic acid, carboxylate, sulfonic acid, sulfonate, hydrogen sulfate, and sulfate. 
     
     
         25 . The compound of  claim 17 , wherein R comprises a group consisting of amino, secondary amino, tertiary amino, quaternary amino, azaryl, hydroxyaryl, carboxylic acid, carboxylate, sulfonic acid, sulfonate, hydrogensulfate, and sulfate. 
     
     
         26 . The compound of  claim 17 , wherein R comprises a group selected from the group consisting of a poly(ethylene glycol) moiety, a poly(propylene glycol) moiety, a linear oligo- or polysaccharide moiety, a branched oligo- or polysaccharide moiety, and a cyclic oligo- or polysaccharide moiety. 
     
     
         27 - 28 . (canceled) 
     
     
         29 . A method for establishing the shape of a pH gradient between an anode and a cathode across an electrophoretic device in an isoelectric focusing or isoelectric trapping experiment, comprising
 (a) introducing one or more pI markers having a known pI value into an electrophoretic device, wherein the one or more pI markers comprises a compound of  claim 17 ; and   (b) applying an electric field sufficient for a period of time sufficient to separate and concentrate the one or more pI markers.   
     
     
         30 . The method of  claim 29  further comprising determining the position of the separated and concentrated pI markers in the electrophoretic device, and plotting their pI values as a function of their position in the electrophoretic device thereby establishing the shape of pH gradient in the device. 
     
     
         31 . The method of  claim 30  further comprising introducing one or more ampholytic analytes into the electrophoretic device prior to or after applying the electric field, wherein applying the electric field is effective to separate and concentrate the one or more pI markers and the one or more ampholytic analytes. 
     
     
         32 . The method of  claim 31  further comprising determining the position of the separated and concentrated ampholytic analytes in the electrophoretic device, thereby establishing the pIs of the one or more ampholytic analytes. 
     
     
         33 . The method of  claim 30  further comprising introducing one or more ampholytic analytes into the electrophoretic device and applying an electric field sufficient for a period of time sufficient to separate and concentrate the one or more ampholytic analytes. 
     
     
         34 . The method of  claim 33  further comprising determining the position of the separated and concentrated ampholytic analytes in the electrophoretic device, thereby establishing the pIs of the one or more ampholytic analytes. 
     
     
         35 . The method of  claim 29 , wherein the electrophoretic device is an isoelectric focusing device utilizing a suitable mixture of carrier ampholytes, an immobilized pH gradient device or a combination of the two, or an isoelectric trapping device utilizing a suitable series of ion-permeable buffering media, in the absence or presence of carrier ampholytes or isoelectric buffers. 
     
     
         36 - 46 . (canceled)