Immobilized Iminobiotin on Magnetic Poly (Vinyl Alcohol) Microspheres for Single-step Purification of Streptavidin

Figures & data

Figure 1 Scheme of activation and immobilization of MPVAMS with NHS-iminobiotin.

Figure 2 ESEM micrographs of MPVAMS with an average diameter from 100 to 200 µm.

Figure 3 XPS spectra of MPVAMS before (A) and after activation and NHS-iminobiotin immobilization (B).

Figure 4 Effect of different kinds of spacers on the absorbance of streptavidin by iminobiotin-MPVAMS Set conditions: amount of NHS-iminobiotin: 5 mg/mL MPVAMS; amount of supernatant: 10 mg of total protein; adsorption time: 45 min; elution: 0.1 M acetic acid for 30 min.

Figure 5 Effect of NHS-iminobiotin added on the absorbance of streptavidin by iminobiotin-MPVAMS. Set conditions: spacer: EDA; supernatant amount: 10 mg of total protein; adsorption time: 45 min; elution: 0.1 M acetic acid for 30 min.

Figure 6 Effect of amount of supernatant on the absorbance of streptavidin by iminobiotin-MPVAMS. Set conditions: spacer: EDA; amount of NHS-iminobiotin: 5 mg/mL MPVAMS; adsorption time: 45 min; elution: 0.1 M acetic acid for 30 min.

Figure 7 Effect of adsorption time on the absorbance of streptavidin onto iminobiotin-MPVAMS. Set conditions: spacer: EDA; amount of NHS-iminobiotin: 5 mg/mL MPVAMS; amount of supernatant: 10 mg of total protein; elution: 0.1 M acetic acid for 30 min.

Table 1 Purification of Streptavidin by iminobiotin-MPVAMS from culture supernatant^a

	Protein^b (mg)	Streptavidin^c (mg)	Recovery (%)	Purification factor
Supernatant	9.794 ± 0.105	0.250 ± 0.013	–	1
Purified product	0.235 ± 0.006	0.225 ± 0.007	90.15	38

^aAll determinations were repeated three times by 1 mL of iminobiotin-MPVAMS.

^bThe value of protein was determined by Lowry Method (mean value ± SD).

^cStreptavidin was evaluated by sandwich ELISA (mean value ± SD).

Figure 8 (A) HPSEC and (B) SDS-PAGE and (C) Western blot analysis of purified streptavidin by iminobiotin-MPVAMS.