6. Collect the flow-through fraction (FT).
7. Wash the column three times, each wash containing one CV
(5 mL) of buffer C and collect as W1, W2, and W3 fractions,
respectively.
8. Elute the bound material (excised fluorescent iRATmC segment, TEV-His 6 and contaminating proteins) from the column
by adding three CVs (15 mL) of buffer D and collect as the E
fraction.
9. Measure the protein concentration of each fraction using a
NanoDrop spectrophotometer.
10. Mix the FT and W1 fractions and concentrate the protein using
an Amicon Ultra-15 (MWCO 30 K) centrifugal concentrator
to a volume of 5 mL.
11. (SEC) Inject the 5 mL of concentrated protein sample onto a
HiLoad16/60 Superdex200 column pre-equilibrated with
TBS at a flow rate of 1 mL/min.
12. Day 3: Collect 2 mL fractions. Analyze the UV trace, and pool
the peak fractions.
13. Perform SDS-PAGE to check the purity.
14. Measure the protein concentration of each fraction using a
NanoDrop spectrophotometer to estimate the protein yield.
15. Use an aliquot to evaluate the biophysical and/or functional
properties of the purified tag-free Fab fragment (Fig. 6 as a
typical example). Ideally, binding assays, such as SPR, ITC, or
ELISA, should provide valuable information.
16. Concentrate the remaining purified Fab fragment using a Amicon Ultra-15 (MWCO 10 K) centrifugal concentrator to an
appropriate protein concentration (e.g., 10 mg/mL).
17. Re-measure the protein concentration of each fraction using a
NanoDrop spectrophotometer.
18. Prepare 100 μL aliquots, and flash-freeze with liquid nitrogen.
19. Store at À80
C.
4 Notes
1. Antibody sequencing from monoclonal hybridoma cell lines
can be performed as described previously [14]. Amino acid
sequence data for the known therapeutic antibodies can be
retrieved from DrugBank (http://www.drug-bank.ca/).
2. The TEV-His 6 was prepared as described previously [15]. It is
not necessary to add any additional DTT to the TEV-His 6 from
that already contained in its storage buffer.
100
Norimichi Nomura et al.
7. Wash the column three times, each wash containing one CV
(5 mL) of buffer C and collect as W1, W2, and W3 fractions,
respectively.
8. Elute the bound material (excised fluorescent iRATmC segment, TEV-His 6 and contaminating proteins) from the column
by adding three CVs (15 mL) of buffer D and collect as the E
fraction.
9. Measure the protein concentration of each fraction using a
NanoDrop spectrophotometer.
10. Mix the FT and W1 fractions and concentrate the protein using
an Amicon Ultra-15 (MWCO 30 K) centrifugal concentrator
to a volume of 5 mL.
11. (SEC) Inject the 5 mL of concentrated protein sample onto a
HiLoad16/60 Superdex200 column pre-equilibrated with
TBS at a flow rate of 1 mL/min.
12. Day 3: Collect 2 mL fractions. Analyze the UV trace, and pool
the peak fractions.
13. Perform SDS-PAGE to check the purity.
14. Measure the protein concentration of each fraction using a
NanoDrop spectrophotometer to estimate the protein yield.
15. Use an aliquot to evaluate the biophysical and/or functional
properties of the purified tag-free Fab fragment (Fig. 6 as a
typical example). Ideally, binding assays, such as SPR, ITC, or
ELISA, should provide valuable information.
16. Concentrate the remaining purified Fab fragment using a Amicon Ultra-15 (MWCO 10 K) centrifugal concentrator to an
appropriate protein concentration (e.g., 10 mg/mL).
17. Re-measure the protein concentration of each fraction using a
NanoDrop spectrophotometer.
18. Prepare 100 μL aliquots, and flash-freeze with liquid nitrogen.
19. Store at À80
C.
4 Notes
1. Antibody sequencing from monoclonal hybridoma cell lines
can be performed as described previously [14]. Amino acid
sequence data for the known therapeutic antibodies can be
retrieved from DrugBank (http://www.drug-bank.ca/).
2. The TEV-His 6 was prepared as described previously [15]. It is
not necessary to add any additional DTT to the TEV-His 6 from
that already contained in its storage buffer.
100
Norimichi Nomura et al.