Catalogue Number: 3009-AAT
|Shelf Life:||12 months|
Description: Biotin-X nitrilotriacetic acid (biotin-X NTA) is widely used to detect histidine-tagged proteins immobilized on nitrocellulose membranes. The NTA moiety of biotin-X NTA chelates Ni ion that is also chelated with histidine tags. The NTA-polyHis-complex can be detected using standard enzyme-linked streptavidin methods. Biotin-X NTA can be used to detect less than 0.1 pmol of histidine-tagged protein using a streptavidin—horseradish peroxidase conjugate and chemiluminescence techniques. Biotin-X NTA can be removed from the histidine-tagged protein at pH 4.8, allowing the blot to be reanalyzed with another probe. In combination with fluorescent avidin conjugates, this NTA biotin derivative can be used for detecting polyhistidine-containing biomolecules such as fusion proteins. This NTA Biotin derivative is a bifunctional reagent that is used to detect histidine-tagged proteins immobilized. 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.