Avidin affinity chromatography

FIGURE 15.3 Outline of experimental protocol used for ICAT differential protein expression profiling. Protein mixtures from two cell populations are labeled with light or heavy isotopic versions of a cleavable ICAT reagent. Labeled proteins are combined, subject to multidimensional separation by SCX, RP, and avidin affinity chromatography, then analyzed by tandem MS for peptide and protein identification. Based on the relative ratio of the two isotopically labeled peptides, a relative abundance of protein expression can be determined. 

Exterrsion of the above procedure trsing ethanedithiol, enabling subsequent biotirtylation to obtain a phosphoprotein isotope-coded affinity tag (PhlAT) [53-54]. This errables phosphopeptide isolation by avidin affinity chromatography. 

Biotin-labeled glycan tf HRP-avidin Affinity chromatography 16... 

The tagged cysteine-containing fragments are isolated by avidin affinity chromatography. 

The combined sample is digested and the modified peptides are recovered using avidin affinity chromatography. 

Avidin (from egg white) [1405-69-2] Mr -70,000. Purified by chromatography of an ammonium acetate soln on CM-cellulose [Green Biochem J 101 774 1966]. Also purified by affinity chromatography on 2-iminobiotin-6-aminohexyl-Sepharose 4B [Orr 7 Bio/C/iew 256 761 1981]. It is a biotin binding protein. 

Isolation of complexed molecules may be done by affinity chromatography using a column of immobilized avidin or immobilized streptavidin. Cleavage of the disulfide bond of the crosslinker may be done by treatment with 50 mM dithiothreitol (DTT). For additional information on the use of sulfo-SBED in the study of protein interactions, see Chapter 28, Section 3.1. 

In another example, ligands can be biotinylated with a cleavable biotinylation reagent and then incubated with receptor molecules. The resulting complex can be isolated by affinity chromatography on immobilized (strept)avidin. Final purification of the ligand-receptor can be accomplished by cleaving the biotin modification sites while the complex is still bound to the support. The receptor complex thus can be eluted from the column without the usual harsh conditions required to break the avidin-biotin interaction. 

Cuatrecasas, P., and Wilchek, M. (1968) Single-step purification of avidin from egg white by affinity chromatography of biocytin-Sepharose columns. Biochem. Biophys. Res. Comm. 33, 235-246. 

For affinity chromatography, avidin or streptavidin are coupled to supports using Protocol 3.6.2. Biotin is covalently bound to proteins or other primary amino groups bearing molecules using its N-hydroxsuccinimide ester. ... 

The pH-dependent binding of 2-imino-biotin (Fig. 3g) has been used in affinity chromatography to purify strept(avidin) because binding is easily reversible [85]. Specifically,bin ding occurs under alkaline conditions (pH 10) and release occurs under acidic conditions (pH 4). An amine-reactive derivative of desthiobiotin (Fig. 3h), a non-sulfur containing metabolic precursor to biotin that is bound to strept(avidin) with lower affinity than biotin, has been used to form reversible complexes with strept(avidin) [74]. Release is achieved by incubation in the presence of free desthiobiotin or biotin. Recently developed lower affinity derivatives include 9-methyl biotins [86]. Another approach covalently attaches a photocleavable biotin derivative (Fig. 3h) to molecules that are released following exposure to 200 nm light [75]. 

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