Pierce Monomeric Avidin Resins and Kit

Pierce Monomeric Avidin Kit and Resins enable gentle, non-denaturing affinity purification of biotinylated molecules. The Immobilized Monomeric Avidin protein binds biotin with high specificity and moderate affinity, allowing non-biotin molecules to be washed away and then the bound biotin-labeled molecules to be competitively eluted using 2 mM biotin in phosphate buffered saline (PBS). This technique provides the gentlest elution conditions for biotinylated protein purification and avoids the contamination and other problems associated with traditional avidin and streptavidin methods. The monomeric avidin column can then be regenerated by using 0.1 M glycine to strip the column of residual bound biotin without losing the ability to bind another biotinylated sample.

With typical avidin or streptavidin, the biotin-binding affinity (Kd = 10^-15 M) is so great that purification with these traditional media require denaturing conditions for elution, such as 8 M Guanidine•HCl at pH 1.5 or boiling in reducing SDS-PAGE sample loading buffer. In addition to their adverse effects on the biotinylated protein of interest, such harsh elution conditions typically cause extraneous proteins (i.e., the avidin or streptavidin subunits) to strip from the resin and co-elute with the desired purification product. By contrast, when avidin is coupled to a resin (e.g., crosslinked beaded agarose or UltraLink Resin) as the subunit monomer, its specificity for biotin is retained, but its biotin-binding affinity decreases to a level (Kd = 10^-8 M ) that is conducive to subsequent elution.

Highlights: Non-denaturing – purifies biotinylated products using mild elution conditions (2 mM free biotin) Reusable – Monomeric Avidin Agarose can be regenerated and reused at least 10 times, with only a marginal loss in biotin binding capacity (approximately 2.5% decrease per regeneration) Specific – retains biotin-binding specificity and low nonspecific binding of native avidin Flexible – bind samples in a variety of physiological buffers and elute either with 0.1 M glycine or by competition with 2 mM biotin Good binding capacity – greater than 1.2 mg biotinylated BSA/ml resin Convenient – purchase standalone agarose or UltraLink Resins or a complete purification kit with prepacked column and buffers

Biotin binding and elution with Monomeric Avidin Agarose

References:

1. Petrotchenko E. V. et al. (2011) An isotopically coded cid-cleavable biotinylated cross-linker for structural proteomics. Mol. Cell. Proteomics. 10, M110.001420-.
2. Friis S. et al. (2011) Transport via the transcytotic pathway makes prostasin available as a substrate for matriptase. J. Biol. Chem. 286, 5793-802.
3. Nie Y. and Kaback H. R. (2010) Sugar binding induces the same global conformational change in purified lacy as in the native bacterial membrane. PNAS. 107, 9903-8.
4. Forejtnikova H. et al. (2010) Transferrin receptor 2 is a component of the erythropoietin receptor complex and is required for efficient erythropoiesis. Blood. 116, 5357-67.
5. Feria Bourrellier A. B. et al. (2010) Chloroplast acetyl-coa carboxylase activity is 2-oxoglutarate-regulated by interaction of pii with the biotin carboxyl carrier subunit. PNAS. 107, 502-7.

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