Biology Reference
In-Depth Information
13. For Golgi enrichment, it is not recommended to use a sucrose
concentration less than 1.0 M (gradient 1). However, if the
intention is to enrich for the ER and other secretory compart-
ments at the expense of the Golgi, gradient 2 can be made
using a slightly lower concentration (e.g., 0.85-0.95 M gradi-
ent buffer 2) and initial spin speed of 100,000 ×
g
rather than
60,000 ×
g
(gradient 1) can be used.
14. Of the two bands present in gradient 1, the lower should be
thicker than the upper. A distinctly formed yellow-white band
1-3 mm thick (in both gradients 1 and 2) will give optimized
results obtained after FFE.
15. Ideally,
steps 1
and
2
(Subheading
3.2
) should be completed as
fast as practicably possible. Tubes containing gradient buffer 1
can be prepared in advance. Setup of the FFE and stabilization
of the current should have been completed so as to coincide with
step 9
. The current normally takes not more than 20-30 min to
stabilize. The conductivity may increase as the sample buffer
enters the chamber. This can be rectifi ed by lowering the voltage
by, e.g., 20-30 V and waiting for approximately 5 min before
starting to collect samples.
16. This is a nontrivial task and some previous experience or
instructions in the setup of the system for ZE-FFE has been
assumed. Use of fresh fi lters, membranes, and electrode gas-
kets can improve separation performance considerably. Great
care should be taken that the plastic spacer is exactly centered.
Tubing sections under the peristaltic pumps should be checked
thoroughly for hairline cracks prior to setup and changed if
necessary. The sample inlet tubing should be exchanged if any
kinks are present. Excessive indentation from the spacer or the
inlet tube on the lower, temperature-cooled plate indicates
that a change of plastic cover sheeting is required for optimal
separation. All FFE buffers should be made up precisely to the
stated pH; otherwise the conductivity will be too high and the
current will not stabilize as desired. Much useful material,
including references, is available at http://www.ffeservice.com.
17. Media fl ow rate and voltage are the two principal parameters
that affect sample migration. For different types of sample
preparation a ratio between the two should be optimized;
however below a media fl ow rate of 200 mL/h, diffusion
between streams of subcellular compartments may occur. The
effect of changing parameters on protein distribution can be
instantaneously verifi ed by measuring protein content at
280 nm in UV-transparent 96-well plates.
18. If particulates are present in the sample, pass the sample once
through a glass Pasteur pipette. This improves homogeneity and
seems to improve separation but the effect on compartment
integrity should be checked for each different type of sample.
