Biology Reference
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7. Remove the supernatant such that the cushion surface is
disturbed as little as possible ( see Note 12 ). Onto this surface,
gently layer 15-20 mL of gradient buffer 2. If enriching for
intact Golgi cisternae only, follow this with 8-10 mL of
gradient buffer 3 and 5-8 mL of gradient buffer 4 (gradient 1).
If enriching for generic secretory membranes, follow gradient
buffer 2 with 12-15 mL of gradient buffer 4 (gradient 2), or
until tubes are at least two-thirds full ( see Note 13 ).
8. Ultracentrifuge samples at 100,000 × g for 90 min.
9. For gradient 1, two bands should be present. Discard the
upper band and remove the lower band with a plastic Pasteur
pipette. For gradient 2 a single band should be present
( see Note 14 ).
10. Measure the protein concentration of the collected sample and
dilute so that the sucrose concentration is as close to FFE
buffer 1 as possible, although a total protein concentration less
than 0.75
L is not recommended. Keep the desired band
in a 15 mL tube on ice ( see Note 15 ).
μ
g/
μ
3.3 Separation of
Secretory Membranes
Using ZE-FFE
1. Ensure that the FFE system is correctly set up for ZE-FFE
(Fig. 1 ), the temperature set to 8 °C, the media pump has been
calibrated, and all necessary quality control tests have been
undertaken ( see Note 16 ).
2. Load FFE buffer 1 (inlet tubes 2-6), FFE buffer 2 (inlets 1 and
7), and FFE buffer 3 (electrodes) and slowly fi ll chamber with
buffers avoiding air bubbles. Set the media fl ow rate to
250 mL/h. Switch on electrode circuits; set the voltage,
current, and power to 700 V, 150 mA, and 100 W, respectively;
and switch on the voltage ( see Note 17 ). Allow the system to
equilibrate and stabilize for 20-30 min prior to use.
3. Load the sample at a fl ow rate of 2,500-3,000
L/h. The sample
should appear homogenous and be visible in the lower half of
the separation chamber but is usually too dilute to be seen in the
upper half ( see Note 18 ).
μ
4. After about 10 min, start collecting samples in the precooled
2 mL deep-well plates ( see Note 19 ).
5. Throughout the ZE-FEE process, monitor protein distribution
by removing 150-250
L from plates and measuring at 280 nm
using UV-transparent 96-well plates. Protein peak positions
and profi les should remain constant throughout separation.
6. After the sample has been processed through the FFE, pool
fractions of interest from multiple plates and centrifuge at
100,000 × g for 60 min ( see Note 20 ).
7. Remove the supernatant and rinse pellets gently by trickling
4-5 mL of 10 mM Tris-HCl (pH 7.5) down the sides of tubes.
μ
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