MONOMERIC AVIDIN GEL
from
Thermo Scientific Pierce Protein Research Products
Description
Now there is a more gentle method for purifying biotinylated molecules. In order to break the biotin-avidin interaction, 8 M Guanidine•HCl at pH 1.5 is required. In affinity chromatography techniques with immobilized avidin/streptavidin, the use of Guanidine•HCl may result in denaturation of the biotinylated protein and cause irreversible damage. In addition, the sample will contain extraneous proteins (avidin or streptavidin subunits) that were stripped off the column as a result of the harsh elution conditions. In addition, the leaching off of the avidin or streptavidin subunits will cause the column to lose much of its biotin binding capacity. One method for eluting biotinylated molecules under mild conditions involves the use of an immobilized monomeric avidin support. When avidin is coupled to a gel (agarose bead) as the subunit monomer, the specificity for biotin is retained, but the affinity for biotin binding substantially decreases (Kd approximately 10-8 M ).
Immobilized Monomeric Avidin and the ImmunoPure Immobilized Monomeric Avidin Kit can be used to bind biotinylated molecules, and the bound material then can be competitively eluted using 2 mM biotin in phosphate buffered saline (PBS). This technique provides the gentlest elution conditions without contamination of the avidin subunits or substantial loss of column binding capacity. The monomeric avidin column can then be regenerated by using guanidine hydrochloride to strip the column of bound biotin without losing the ability to bind the next biotinylated protein.
Features
- Purifies biotinylated products under mild elution conditions (2 mM free biotin)
- Can be regenerated and reused at least 10 times, with only a marginal loss in biotin binding capacity (approximately 2.5% decrease per regeneration)
- Exhibits little nonspecific binding (3% or less)
Ideal affinity support for reversibly binding biotinylated proteins.