7. Following sonication, the cells are centrifuged for 20 min at
4000 rpm and 4
C (Sorvall Biofuge Primo R Centrifuge) and
the supernatant is transferred to microcentrifuge tubes.
8. The tubes are then centrifuged at 13,000 rpm (Eppendorf
Centrifuge 5415 D) for 10 min to remove any remaining debris
and the supernatant is collected into a single fresh tube.
9. Protein purification is then carried out with a prepared HisTrap
HP column (GE Healthcare).
10. To prepare the HisTrap column, with a column volume
(CV) of 1 mL, the following steps are performed:
(a) Pass 2 CV of DI water over the column.
(b) Pass 2 CV of 100 mM NiSO 4 over the column.
(c) Pass 2 CV of DI H 2 O over the column.
(d) Pass 2 CV of buffer B over the column.
(e) Pass 2 CV of DI H 2 O over the column.
Add the crude protein extract to the column and
collect 1 mL fractions in a 96-well plate.
(f) Pass 20 mL of buffer A containing 5 mM imidazole
through the column followed by successive 5 mL of buffer
A containing the following concentrations of imidazole:
10, 15, 20, 30, 50, 100, and 250 mM (i.e., buffer B),
followed by an additional 10 mL of buffer B passed over
the column during which fractions were no longer
collected.
11. The fractions collected are then evaluated for protein content
using a plate reader to measure absorbance at 280 nM. Fractions which demonstrate an increase in absorbance, typically
fractions containing ~100 mM imidazole, are then evaluated
for the presence of GFP using SDS PAGE using a 10%
acrylamide gel.
12. After the SDS PAGE is completed, the gel is stained using
Coomassie Blue, then washed with water to visualize protein
bands. Fractions containing the protein of interest with little
contaminants are identified using a protein ladder and then
combined.
13. The combined protein fractions are dialyzed overnight into
PBS at 4
C.
14. The concentration of the protein solution is determined using
the Bradford Assay with a clear polystyrene plate. Five microliters of the extract is mixed with 250 μL of the Bradford
reagent and incubated at room temperature for 20 min. Afterward, the absorbance at 595 nm is measured and compared to a
standard curve containing 0–1 mg/mL of BSA (0, 0.2, 0.4,
0.6, 0.8, and 1.0 mg/mL).
Liposomal Dual Antigen Delivery
481
4000 rpm and 4
C (Sorvall Biofuge Primo R Centrifuge) and
the supernatant is transferred to microcentrifuge tubes.
8. The tubes are then centrifuged at 13,000 rpm (Eppendorf
Centrifuge 5415 D) for 10 min to remove any remaining debris
and the supernatant is collected into a single fresh tube.
9. Protein purification is then carried out with a prepared HisTrap
HP column (GE Healthcare).
10. To prepare the HisTrap column, with a column volume
(CV) of 1 mL, the following steps are performed:
(a) Pass 2 CV of DI water over the column.
(b) Pass 2 CV of 100 mM NiSO 4 over the column.
(c) Pass 2 CV of DI H 2 O over the column.
(d) Pass 2 CV of buffer B over the column.
(e) Pass 2 CV of DI H 2 O over the column.
Add the crude protein extract to the column and
collect 1 mL fractions in a 96-well plate.
(f) Pass 20 mL of buffer A containing 5 mM imidazole
through the column followed by successive 5 mL of buffer
A containing the following concentrations of imidazole:
10, 15, 20, 30, 50, 100, and 250 mM (i.e., buffer B),
followed by an additional 10 mL of buffer B passed over
the column during which fractions were no longer
collected.
11. The fractions collected are then evaluated for protein content
using a plate reader to measure absorbance at 280 nM. Fractions which demonstrate an increase in absorbance, typically
fractions containing ~100 mM imidazole, are then evaluated
for the presence of GFP using SDS PAGE using a 10%
acrylamide gel.
12. After the SDS PAGE is completed, the gel is stained using
Coomassie Blue, then washed with water to visualize protein
bands. Fractions containing the protein of interest with little
contaminants are identified using a protein ladder and then
combined.
13. The combined protein fractions are dialyzed overnight into
PBS at 4
C.
14. The concentration of the protein solution is determined using
the Bradford Assay with a clear polystyrene plate. Five microliters of the extract is mixed with 250 μL of the Bradford
reagent and incubated at room temperature for 20 min. Afterward, the absorbance at 595 nm is measured and compared to a
standard curve containing 0–1 mg/mL of BSA (0, 0.2, 0.4,
0.6, 0.8, and 1.0 mg/mL).
Liposomal Dual Antigen Delivery
481