Single-step synthesis and characterization of biotinylated nitrilotriacetic acid, a unique reagent for the detection of histidine-tagged proteins immobilized on nitrocellulose.
Using a one-step reaction, a bifunctional compound was synthesized for detecting histidine-tagged proteins immobilized on nitrocellulose. This compound has a biotin as one functional group and a nitrilotriacetic acid as the other. The nitrilotriacetic acid is used to chelate a Ni(II) ion at four of its six coordination sites. The remaining two sites are available for binding to a histidine tag. The biotin functional group can then be detected using a streptavidin-horseradish peroxidase conjugate and chemiluminescence. Using this biotinylated nitrilotriacetic acid, it is possible to detect less than 0.11 pmol of histidine-tagged Escherichia coli RNA polymerase sigma70 subunit. This reagent is also able to specifically detect His-tagged sigma70 from a whole cell lysate following SDS-PAGE and transfer to nitrocellulose. The reagent can be dissociated from the His-tagged protein at pH 4.8 and the blot can be reprobed with a monoclonal antibody for detection of different proteins on the same blot.
McMahan
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McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 53706, USA.
Burgess
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D001710:Biotin / Q000031:analogs & derivatives*; D015153:Blotting, Western / Q000379:methods*; D003101:Collodion / Q000737:chemistry; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry; D009532:Nickel / Q000737:chemistry; D009571:Nitrilotriacetic Acid; D011994:Recombinant Proteins / Q000032:analysis*; D012441:Saccharomyces cerevisiae; D012808:Sigma Factor / Q000032:analysis
D011994:Recombinant Proteins; D012808:Sigma Factor; D006639:Histidine; D001710:Biotin; D009532:Nickel; D003101:Collodion; C013411:biocytin; D008239:Lysine; D009571:Nitrilotriacetic Acid