Biology Reference
In-Depth Information
8. TGS buffer is normally made as a 10× stock solution. First,
make 1 L of 10× TGS buffer consisting of 30.3 g Tris, 141.4 g
glycine, and 10 g SDS. Just before use, dilute 100 mL of 10×
TGS buffer with 900 mL of water.
9. Unpolymerized acrylamide is neurotoxic. Acrylamide powder
requires careful handling. Wear gloves, a clean lab coat, and a
mask, and pay attention to people around you when weighing
acrylamide. Store at 4 °C. Add polymerization agent before
discarding any spare acrylamide solution.
10. These solutions should be dispensed into a small volume and
sealed with parafi lm to prevent contamination and evapora-
tion. Store at 4 °C and use within 1 month of preparation.
11. DTT and IAA can be easily modifi ed in solution for a short
period and TFA evaporates quickly. Solutions including DTT,
IAA, and TFA should be freshly prepared just immediately
before use.
12. For
Arabidopsis
PM preparation, plants must be put in homog-
enizing medium directly and immediately after harvest and
washing. Subsequently, plants should be cut with clean scissors
in the medium.
13. For
Arabidopsis
PM preparation, the homogenates should be
centrifuged at 5,000 ×
g
for 10 min.
14. In this step, homogenization should not be too long or too
vigorous because harsh homogenization can severely disrupt
membrane integrity.
15. Two-phase partitioning is the most important step for prepar-
ing highly purifi ed PM. When the upper phase of the two-
phase partition medium is removed, the Pasteur pipette should
be moved from left to right near the boundary of the two
phases to prevent taking lower phase. For
Arabidopsis
PM
preparation, the two phases should be centrifuged at 440 ×
g
for 5 min.
16. The yield of the PM preparation is expected to be 2.5 mg pro-
tein from 70 to 100 g (FW) of oat leaves.
17. One of the keys to making a good step gradient with sucrose
solutions is pouring the solution slowly along the inner wall of
the tube.
18. In this step, the upper portions of the white band should be
discarded fi rst and then the DRM layer should be collected
carefully.
19. All parts of the gel cassette should be wiped with ethanol or
acetone on cleaning tissue to prevent contamination with
other proteins including keratin.
