Table 1.

Binding of biotinylated BSA to M18-L-DBD matrix, M18-L-ST·SC-L-DBD matrix and M18-Affigel.

SAV	Matrix	Coupling method	SAV coupled (µg)	SAV coupled (nmole)	Biotin binding site (nmole)	BSA binding capacity (µg)	BSA binding capacity (nmole)	nmole BSA/ nmole SAV	Capture efficiency (%)
M18-L-DBD (Fully saturated matrix)	Sephadex	biocoupling	708 ± 13*	5	20	485 ± 3*	7.30	1.46	73.0
M18-L-DBD (Half saturated matrix)	Sephadex	biocoupling	354	2.5	10	252 ± 2.9*	3.79	1.52	75.8
M18-L-ST·SC-L-DBD (Fully saturated matrix)	Sephadex	biocoupling	610 ± 6*	2.85	11.4	NA	NA	NA	NA
M18-L-ST·SC-L-DBD (Half saturated matrix)	Sephadex	biocoupling	305	1.43	5.72	143 ± 2.0*	2.15	1.50	75.2
M18	Affi-gel	Chemical coupling	1,000	15.1	60.4	981 ± 5.0*	14.77	0.98	48.9
Recombinant SAV (Commercial)	Sepharose CL-6B	Chemical coupling	239–318**	4.5–6**	18–24	NA	NA	NA	NA

Table 1. 1-ml columns containing one of the affinity matrices were overloaded with biotinylated BSA. For each matrix, the amount in flow-through and wash fractions containing biotinylated BSA was quantitated by Bio-Rad Protein Assay Dye Reagent. The amount captured was estimated as the balance between the amount loaded and the amount in the flow through plus wash fractions. BSA used in this study has 12 biotin moieties per protein. Number of biotin binding site in each column is calculated by the number (nmoles) of M18 (and its derivatives) immobilized to the matrix × 4 since each streptavidin has four biotin binding sites. Capture efficiency is determined based on the assumption that one tetrameric streptavidin can bind two biotinylated BSA proteins. The molecular weight for the monomer of M18, M18-L-DBD, M18-L-ST·SC-L-DBD, recombinant streptavidin (commercial) and BSA is 16,519, 35,038, 53,426, 13,250 and 66,430, respectively. * indicates that the value represents an average of three trials. Data are expressed as average ± SD. ** indicates that the values were estimated based on the number of biotin binding site. NA: data not available.