700 mL, and adjust pH with HCl or NaOH if required, make up to
1 L with deionized water, and store at 4
C until use (see Note 1).
2.3 Ammonium
Sulfate Precipitation
Ammonium sulfate, magnetic stirrer, and glass beakers.
2.4 Dialysis
50 mM Na-phosphate buffer (pH 7.0).
Dialysis cellulose membrane.
2.5 Gel Filtration
Sephadex G-25 media packed in glass column (3 Â 30 cm).
Running buffer: 50 mM Na-phosphate buffer (pH 7.0).
Test tubes for the collection of fractions.
2.6 Ion-Exchange
Chromatography
DEAE-sephadex in glass column (3 Â 30 cm).
Column buffer: 50 mM Na-phosphate buffer (pH 7.0).
Running buffer: 0.1, 0.15, 0.2, and 0.25 M NaCl in column buffer
(50 mM Na-phosphate).
Test tubes for the collection of fractions.
3 Methods
Perform all experiments at low light/dark and at 4
C to prevent
PC denaturation, unless otherwise specified.
A scheme of the overall purification process is shown in Fig. 1.
3.1 Crude Extract
Preparation
1. Weight 100 mg of lyophilized/dry cell mass of A. platensis into
a centrifuge tube.
2. Add 10 mL of ice-cold Na-phosphate buffer to the biomass
and vortex for 30 s.
3. Sonicate the suspension on the ice at a frequency of 20 kHz for
2 min (9 cycles, 50% power).
4. Centrifuge at 6,000 Â g for 15 min at 4
C to remove the cell
debris.
5. Transfer the supernatant to a clean beaker (see Notes 2 and 3).
3.2 Ammonium
Sulfate Precipitation
and Dialysis
1. Place the beaker containing the crude phycocyanin extract on
the ice (this can be done by placing crude extract containing
beaker in an another larger beaker containing crushed ice) and
stir with a magnetic stirrer.
2. While stirring slowly, add a pre-weighed quantity of ammonium sulfate to reach a saturation of 30%, and stir the mixture
for nearly 2 h (see Notes 4 and 5).
3. Transfer the ammonium sulfate precipitated solution to conical
centrifuge tubes and centrifuge at 6,000 Â g for 15 min at 4
C.
4. Collect the supernatant and discard the pellet.
Phycocyanin Purification
175
1 L with deionized water, and store at 4
C until use (see Note 1).
2.3 Ammonium
Sulfate Precipitation
Ammonium sulfate, magnetic stirrer, and glass beakers.
2.4 Dialysis
50 mM Na-phosphate buffer (pH 7.0).
Dialysis cellulose membrane.
2.5 Gel Filtration
Sephadex G-25 media packed in glass column (3 Â 30 cm).
Running buffer: 50 mM Na-phosphate buffer (pH 7.0).
Test tubes for the collection of fractions.
2.6 Ion-Exchange
Chromatography
DEAE-sephadex in glass column (3 Â 30 cm).
Column buffer: 50 mM Na-phosphate buffer (pH 7.0).
Running buffer: 0.1, 0.15, 0.2, and 0.25 M NaCl in column buffer
(50 mM Na-phosphate).
Test tubes for the collection of fractions.
3 Methods
Perform all experiments at low light/dark and at 4
C to prevent
PC denaturation, unless otherwise specified.
A scheme of the overall purification process is shown in Fig. 1.
3.1 Crude Extract
Preparation
1. Weight 100 mg of lyophilized/dry cell mass of A. platensis into
a centrifuge tube.
2. Add 10 mL of ice-cold Na-phosphate buffer to the biomass
and vortex for 30 s.
3. Sonicate the suspension on the ice at a frequency of 20 kHz for
2 min (9 cycles, 50% power).
4. Centrifuge at 6,000 Â g for 15 min at 4
C to remove the cell
debris.
5. Transfer the supernatant to a clean beaker (see Notes 2 and 3).
3.2 Ammonium
Sulfate Precipitation
and Dialysis
1. Place the beaker containing the crude phycocyanin extract on
the ice (this can be done by placing crude extract containing
beaker in an another larger beaker containing crushed ice) and
stir with a magnetic stirrer.
2. While stirring slowly, add a pre-weighed quantity of ammonium sulfate to reach a saturation of 30%, and stir the mixture
for nearly 2 h (see Notes 4 and 5).
3. Transfer the ammonium sulfate precipitated solution to conical
centrifuge tubes and centrifuge at 6,000 Â g for 15 min at 4
C.
4. Collect the supernatant and discard the pellet.
Phycocyanin Purification
175