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 tag 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 inability 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, allows 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 complex, for the detection and quantitation of hexa-histidine-tagged recombinant proteins in solution. The surface plasmon resonance technique also allows for the oriented immobilization of the recombinant proteins for subsequent ligand interaction studies.
O'Shannessy
D J
DJ
SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
O'Donnell
K C
KC
Martin
J
J
Brigham-Burke
M
M
D000345:Affinity Labels; D015374:Biosensing Techniques; D015153:Blotting, Western / Q000379:methods*; D005069:Evaluation Studies as Topic; D006639:Histidine / Q000737:chemistry; D007202:Indicators and Reagents; D008024:Ligands; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry
D000345:Affinity Labels; D007202:Indicators and Reagents; D008024:Ligands; D011994:Recombinant Proteins; D006639:Histidine