Biology Reference
In-Depth Information
5.
Reswelling tray (GE Healthcare).
6.
Paper wicks (GE Healthcare).
7.
Loading cups (GE Healthcare).
8.
Ceramics IPG strip tray (GE Healthcare).
9.
DTT equilibration solution: dissolve 0.05 g of DTT and 1.8 g
urea in 5 mL of proprietary IPG equilibration buffer (Serva,
Heidelberg, Germany). This recipe is designed for two strips.
Adjust the amount and volume according to the number of
strips.
10. Iodoacetamide (IAA) equilibration solution: dissolve 0.125 g
IAA and 1.8 g of urea in 5 mL IPG equilibration buffer (Serva).
Add a suffi cient volume of saturated bromophenol blue solu-
tion until the IAA equilibration solution is a deep blue color
and no longer transparent. This ensures that the dye front will
be visible during electrophoretic separation. This recipe is
designed for two strips. Adjust the amount and volume accord-
ing to the number of strips.
11. Anode buffer: dissolve the content of the anode buffer bag
(Serva) in 7.5 L of water.
12. Cathode buffer: dissolve the powder in the cathode buffer bag
(Serva) in 5 L of water.
13. Flat cassettes 2D (Gel Company).
14. 2DGel DALT 12.5% NF (Serva). Store at 4°C until required.
15. ECL Plex rainbow fl uorescent markers (GE Healthcare). Store
at −20°C.
16. Reference markers.
17. Low-melt agarose: weigh 1 g of low-melt agarose (Quantum
Scientifi c, Queensland, Australia) and dissolve in 100 mL of
cathode buffer (see Subheading
2.4
, item 12) on a heated
magnetic stirrer (70°C). Once dissolved, store at room
temperature.
18. IPG strip pusher (Gel Company).
19. Ettan DALT12 electrophoresis system (GE Healthcare).
20. Ettan DIGE Imager (GE Healthcare).
21. DeCyder 2D (v 7.0; GE Healthcare).
2.5. Proteolytic
Digestion for Protein
Identifi cation
1.
Ettan spot cutting robot (GE Healthcare).
2.
Polypropylene 96-well plates with silicone sealing mats
(Axygen, Union City, CA, USA).
3.
100 mM NH
4
HCO
3
stock solution, prepared in water on the
same day as required.
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