SEC-MALS was used to characterize
the recombinant expressed SF
mutants in solutions relating to their purity, native oligomers or
aggregates and molar masses. Analyses were performed on an LC20 prominence
HPLC system equipped with the refractive index detector RID-10A, the
photodiode array detector SPD-M20A (all from Shimadzu, Japan), and
a MALS Heleos Dawn8C plus QELS detector (Wyatt Technology, U.S.A.).
A Superdex 200 10/300 GL column (Cytiva, U.S.A.) was used and equilibrated
with PBS plus 200 mM NaCl (pH 7.4) as running buffer. Experiments
were performed at a flow rate of 0.75 mL min−1 at
25 °C and analyzed using the ASTRA 6 software (Wyatt Technology,
U.S.A.). Proper performance of the MALS was verified by the determination
of the molar mass of a sample of bovine serum albumin. Prior to analysis,
samples were thawed, centrifuged (16000 g, 10 min), and filtered by
a 0.1 μm Ultrafree-MC filter (Merck Millipore, Germany). A total
amount of 25 μg of the respective SF variant was injected for
each measurement.
the recombinant expressed SF
mutants in solutions relating to their purity, native oligomers or
aggregates and molar masses. Analyses were performed on an LC20 prominence
HPLC system equipped with the refractive index detector RID-10A, the
photodiode array detector SPD-M20A (all from Shimadzu, Japan), and
a MALS Heleos Dawn8C plus QELS detector (Wyatt Technology, U.S.A.).
A Superdex 200 10/300 GL column (Cytiva, U.S.A.) was used and equilibrated
with PBS plus 200 mM NaCl (pH 7.4) as running buffer. Experiments
were performed at a flow rate of 0.75 mL min−1 at
25 °C and analyzed using the ASTRA 6 software (Wyatt Technology,
U.S.A.). Proper performance of the MALS was verified by the determination
of the molar mass of a sample of bovine serum albumin. Prior to analysis,
samples were thawed, centrifuged (16000 g, 10 min), and filtered by
a 0.1 μm Ultrafree-MC filter (Merck Millipore, Germany). A total
amount of 25 μg of the respective SF variant was injected for
each measurement.