DETECTION AND QUANTITATION OF HEXA-HISTIDINE-TAGGED RECOMBINANT PROTEINS ON WESTERN BLOTS AND BY A SURFACE-PLASMON RESONANCE BIOSENSOR TECHNIQUE

The use of short peptide affinity tag sequences has become commonplace for the expression and purification of recombinant proteins. Many of these tags are antibody epitopes and detection of tagged proteins via Western blots is straightforward. However, the most common affinity ta g used at present for the expression of recombinant proteins is a hexa histidine, or like sequence, which exhibits strong affinity for Ni(II ). The one drawback of histidine containing affinity tags is the inabi lity to specifically detect such recombinant proteins on Western blots . Here we describe the synthesis and use of biotinyl-nitrilotriacetic acid which, in combination with streptavidin-horseradish peroxidase, a llows for the detection of hexa-histidine-tagged recombinant proteins on Western blots. In addition, we describe a surface plasmon resonance technique, employing a solid-phase Ni(II)-nitrilotriacetic acid compl ex, for the detection and quantitation of hexahistidine-tagged recombi nant proteins in solution. The surface plasmon resonance technique als o allows for the oriented immobilization of the recombinant proteins f or subsequent ligand interaction studies. (C) 1995 Academic Press, Inc .