Click-&-Go® Dde Protein Enrichment Kit
*for enrichment of azide-modified proteins*
Click-&-Go® Dde Protein Enrichment Kit provides all the necessary reagents to perform enrichment of azide-modified proteins through conventional biotin-streptavidin affinity purification. The kit includes a cleavable Dde Biotin Alkyne that allows for release of either captured proteins for intact protein analysis or on-beads digestion followed by the release of peptides for subsequent downstream analysis by mass spectrometry. Captured biomolecules can be released under mild conditions, 2% aqueous hydrazine. Sufficient materials are supplied for 25 enrichments based on the protocol below. The kit provides azide labeled BSA as a positive control.
Extraordinary strength of the streptavidin-biotin interaction allows for efficient capturing of even highly dilute targets; however, it makes recovery of proteins from affinity resins challenging. Conventional methods to elute biotinylated proteins from immobilized avidin include the following: (i) denaturation of streptavidin by boiling the resin in a denaturing buffer that may include high concentrations of chaotropic salts, (ii) trypsin digestion of proteins while they are bound to the resin, or (iii) elution of proteins with excess free biotin. These protocols can co-elute contaminant proteins by releasing nonspecifically bound proteins and/or naturally biotinylated proteins concurrently with labeled proteins. In addition, some of these methods can cause elution of high levels of resin-based peptides along with the proteins of interest, resulting in further sample contamination.
Dde Biotin Alkyne probe eliminates a major limitation of the streptavidin-biotin affinity purification. This reagent contains a biotin moiety linked to an azide (N3) through a spacer arm containing a containing a hydrazine-cleavable Dde moiety. Captured biomolecules can be efficiently released under mild conditions (2% aqueous hydrazine solution), and the small molecular fragment (55.07 Da) left on the labeled protein following the cleavage. These features make the cleavable probe especially attractive for use in biomolecular labeling and proteomic studies.
An alkyne-activated, acid-cleavable biotin probe that allows for elution of the captured biomolecules under mild conditions (5%-10% formic acid).