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Ultrafiltrations with a membrane of 50 kDa MWCO and under different conditions of transmembrane pressures (3 and 5 bar, named in Table 4 as U50-3 and U50-5, respectively) were carried out with genipap extract that was previously microfiltered through the membrane of 0.8 [micro]m.
The solution was dialyzed using dialysis membrane (MWCO 3.5 kDa) and then lyophilized to obtain mPEG-N[H.sub.2].
Note that semipermeable membranes with 3500 g [mol.sup.-1] molecular weight cut-off (MWCO) were chosen in order to prevent the PEG 6000 (i.e., 6000 g [mol.sup.-1] molecular weight) from bypassing the membranes.
Fractions with 280 nm absorbance greater than 5-fold over background were pooled and buffer-exchanged to PBS using Amicon spin filters (50 KDa MWCO, 15 ml; Millipore).
Flat sheet membranes come in the full range of membrane pore sizes, from 0.2[mu] to reverse osmosis porosity of 150 to 100 molecular weight cut-off (MWCO).
The dialysis bag (Sigma, 12000-14000 Da MWCO) was suspended in pH 7.4 phosphate buffer (50 mL) maintained at 37[+ or -]0.5[degrees]C.
Thus, a dialysis membrane with a 14KDa molecular-weight cutoff (MWCO) because it is closest to the desired molecular weight of hyaluronidase .
In the next step the glucan solutions were purified by diafiltration with water (Vivaflow 50 R, MWCO 100,000, Sartorius, Gottingen, Germany) until the conductance of the retentate dropped to 0 [micro]S [cm.sup.-1].
The resulting solution is dialyzed using 10 K MWCO and 0.1-0.5 ml Slide-A-Lyzer cassettes (Thermo Fisher Scientific) spinning under magnetic stirring in 1L of PBS at 4[degrees]C for 5 hours and then overnight after exchanging with fresh PBS.
Before the measurement, the culture supernatants were concentrated from 3 ml to 0.5 ml using Vivaspin 6 columns (10.000 MWCO) (Sartorius, Gottingen, Germany).
Reducing power also varied according to different fractions based on molecular weight cutoff (MWCO).
Urine samples (500 [micro]l) were centrifuged at 17,000g for 10 min at 4[degrees]C and removed from the urinary sediment, to transfer the supernatant into filtration concentrators (100 kDa MWCO, Millipore) and centrifuge it at 4000g for 5-10 min under normal temperature to let almost all solution go through the filters.
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