2. Carefully transfer the supernatant to 1.5 mL Protein LoBind
tube and keep on ice.
3. Assess protein concentration using BCA assay according to
manufacturer’s instruction. Conduct the measurement of the
sample in triplicates.
4. Dilute the sample to a concentration of 1 mg/mL and load
each Amicon
® 30 MWCO with 1 mg of protein according to
BCA readout which is needed for each SEC experiment.
5. Centrifuge at 3200 Â g for 5 min at 4
C. Dilute sample back to
1 mL using ice-cold HNN buffer and gently mix by pipetting
(see Note 4).
6. Repeat step 5 two times for a total of three washes.
7. Centrifuge at 3200 Â g for 4 min (approximately 250 μL left).
8. Centrifuge at 3200 Â g for 5 min (approximately 120/130 μL
left, (see Note 5).
9. Transfer 110 μL to a 1.5 mL Protein LoBind tube being careful
to avoid scratching the filter with the pipette tip.
10. Centrifuge in a to 4
C precooled tabletop centrifuge at
15,000 Â g for 1 min to remove large aggregates.
11. Transfer supernatant to an injection vial and proceed to inject
in the HPLC.
3.4 HPLC Standard
Operation Procedure
Before performing native separations of precious samples, it is
highly recommended to check system performance with an appropriate protein standard mix (see Note 6). For SEC columns which
separate in the range from 15 kDa to 1.5 MDa (e.g., YarraSEC4000), a manufacturer’s standard mix of Bovine Thyroglobin
(670 kDa), IgA (300 kDa), IgF (150 kDa), Ovalbumin (44 kDa),
Myoglobin (17 kDa), and Uridine (244 kDa) is suitable. As a rule
of thumb, with an instrument operating in good/optimal conditions, the Yarra-SEC4000 column separates these standard proteins
with almost base peak resolution. We summarize in Notes 7–9 the
settings that have a noticeable impact on the quality of the native
separation. For the sake of clarity, all steps of native separation
described in this section are performed with a Yarra SEC4000.
HPLC parameters setup: temperature control at 4
C for injection,
column and fractions collector module, flow rate: 0.5 mL/min, UV
detector recording at 260 and 280 nm and collection of 60 fractions
of 125 μL starting from the column void volume (see Note 10) to
the column volume. The fraction volume can be altered if other
SEC settings are applied. Further, for other column materials (see
Table 2) and column sizes, it is recommended to optimize flow rate
and fractionation volume. Specifically, cooled columns can easily
overpressure if operated at a higher flow rate, and thus it is advised
to test the optimal flow rate to avoid column over pressuring.
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Andrea Fossati et al.
tube and keep on ice.
3. Assess protein concentration using BCA assay according to
manufacturer’s instruction. Conduct the measurement of the
sample in triplicates.
4. Dilute the sample to a concentration of 1 mg/mL and load
each Amicon
® 30 MWCO with 1 mg of protein according to
BCA readout which is needed for each SEC experiment.
5. Centrifuge at 3200 Â g for 5 min at 4
C. Dilute sample back to
1 mL using ice-cold HNN buffer and gently mix by pipetting
(see Note 4).
6. Repeat step 5 two times for a total of three washes.
7. Centrifuge at 3200 Â g for 4 min (approximately 250 μL left).
8. Centrifuge at 3200 Â g for 5 min (approximately 120/130 μL
left, (see Note 5).
9. Transfer 110 μL to a 1.5 mL Protein LoBind tube being careful
to avoid scratching the filter with the pipette tip.
10. Centrifuge in a to 4
C precooled tabletop centrifuge at
15,000 Â g for 1 min to remove large aggregates.
11. Transfer supernatant to an injection vial and proceed to inject
in the HPLC.
3.4 HPLC Standard
Operation Procedure
Before performing native separations of precious samples, it is
highly recommended to check system performance with an appropriate protein standard mix (see Note 6). For SEC columns which
separate in the range from 15 kDa to 1.5 MDa (e.g., YarraSEC4000), a manufacturer’s standard mix of Bovine Thyroglobin
(670 kDa), IgA (300 kDa), IgF (150 kDa), Ovalbumin (44 kDa),
Myoglobin (17 kDa), and Uridine (244 kDa) is suitable. As a rule
of thumb, with an instrument operating in good/optimal conditions, the Yarra-SEC4000 column separates these standard proteins
with almost base peak resolution. We summarize in Notes 7–9 the
settings that have a noticeable impact on the quality of the native
separation. For the sake of clarity, all steps of native separation
described in this section are performed with a Yarra SEC4000.
HPLC parameters setup: temperature control at 4
C for injection,
column and fractions collector module, flow rate: 0.5 mL/min, UV
detector recording at 260 and 280 nm and collection of 60 fractions
of 125 μL starting from the column void volume (see Note 10) to
the column volume. The fraction volume can be altered if other
SEC settings are applied. Further, for other column materials (see
Table 2) and column sizes, it is recommended to optimize flow rate
and fractionation volume. Specifically, cooled columns can easily
overpressure if operated at a higher flow rate, and thus it is advised
to test the optimal flow rate to avoid column over pressuring.
278
Andrea Fossati et al.