Biology Reference
In-Depth Information
4
Notes
1. Digitonin is dissolved by heating the solution to 100 °C. After
heating, digitonin can immediately be mixed with other deter-
gents. The rest of the detergent mixture can be stored at
−20 °C. Always reheat solution before usage.
2. Add LDS after the buffer reagents have been weighed (pH ~ 6.8
at room temperature equals pH ~ 7.0 at 4 °C).
3. If larger amounts of buffers are prepared, store at −20 °C.
4. To avoid freeze-thaw cycles of membrane samples, freeze
20
L droplets directly in liquid N
2
. Use an open container
immersed in the liquid N
2
to gather the droplets. For storage
of the droplets, make a hole in the lid of a number of micro
tubes. Place a cardboard stand in liquid N
2
and place the micro
tubes to precool. Ensure that no liquid N
2
fl ows into the tubes.
Gather frozen droplets into the precooled micro tubes using
forceps. Precool the tweezers before picking the droplets.
Store tubes at −80 °C. For experimental access to thylakoid
membranes, stored droplets can be picked separately with for-
ceps and transferred to a fresh tube.
5. Depending on the organism and membrane that should be
solubilized different detergent concentrations and mixtures
need to be tested individually.
6. Use glass evacuation fl asks for degassing. Shake solutions to
initiate gas bubble release. Pay attention that APS and TEMED
are added after degassing.
7. Gradient mixer should be fi lled at least 50 % of each of the
chamber volumes.
8. The chamber next to the tube outlet (chamber 1) has to be
stirred properly whereas stirring of chamber 2 is not necessary.
The stirring bar in chamber 2 acts as balancer for the solution
levels in both chambers.
9. Adjust stirrer speed to maintain solution mixed without aera-
tion. Make sure that no air bubbles block the connection
between chambers.
10. Measure pump speed by determination of time required to
collect a 10 mL fraction.
11. Ensure that the distance between well and separation gel is at
least 0.5 cm.
12. If gels are stored overnight, wrap the gel sandwich in wet tis-
sues and keep combs in place.
13. Depending on the electrophoresis apparatus the gel comb has
to be removed before or after assembly with the glass
sandwich.
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