US 7,070,941 B2 · Methods and compositions for tagging via azido substrates

Abstract

The invention provides methods and compositions for azide tagging of biomolecules. In one embodiment of the invention, proteins are tagged by metabolic incorporation of prenylated azido-analog substrates. Examples of such analogs are azido farnesyl diphosphate and azido farnesyl alcohol. The azido moiety in the resulting modified proteins provides an affinity tag, which can be chemoselectively captured by an azide-specific conjugation reaction, such as the Staudinger reaction, using a phosphine capture reagent. When the capture agent is biotinylated, the resulting conjugates can be detected and affinity-purified by streptavidin-linked- HRP and streptavidin-conjugated agarose beads, respectively. The invention allows detection and isolation of proteins with high yield, high specificity, and low contamination without harsh treatment of proteins.

Claims27

Dependent claims

A method for detecting at least a first isoprenylated protein in a cell comprising:

a) obtaining a substrate of at least a first protein in said cell, wherein the substrate is a synthetic isoprenyl azide substrate comprising at least a first azide;
b) contacting the cell with the synthetic isoprenyl azide substrate under conditions wherein the cell takes up synthetic isoprenyl azide substrate and the synthetic isoprenyl azide substrate reacts with the first protein to produce at least a first isoprenylated protein;
and
c) detecting at least said first isoprenylated protein from proteins produced by said cell by contacting the proteins produced by said cell with a phosphine capture reagent, wherein capture occurs by the Staudinger reaction.

1 more element

Dependencies are inferred from the claim text.

Methods and compositions for tagging via azido substrates

Life of this patent

Methods and compositions for tagging via azido substrates

Life of this patent

Abstract

Claims27