Biology Reference
In-Depth Information
9. Precast gels: Perfect NT gel S 10-20 %, 2-D well, 14 cm × 14 cm
(D.R.C. Coop., Tokyo, Japan).
10. Electrophoresis system.
11. Agarose solution: 0.5 % low-melting agarose. Weigh 0.5 g of
low-melting agarose and add 100 mL of SDS electrophoresis
buffer. Warm to just before bubbling in a microwave. Divide
1 mL of agarose solution into microtubes and store at RT.
12. SDS electrophoresis buffer: 25 mM Tris, 192 mM glycine,
0.1 % (w/v) SDS. Dissolve 4.5 g of Tris base, 21.6 g of glycine,
and 1.5 g of SDS in ultrapure water and top up the volume up
to 1.5 L. In case of frequent use, make a tenfold concentrated
stock solution and store at RT.
13. ECL Plex Fluorescent Rainbow Markers (GE Healthcare).
2.3 Double-Stained
Immunoblotting
1. Tank transfer system.
2. Blotting buffer: 25 mM Tris, 192 mM glycine. Dissolve 15 g
of Tris base and 72 g of glycine in ultrapure water and top up
the volume to 5 L. In case of frequent use, make a tenfold
concentrated stock solution and store at RT.
3. Container: The area of the bottom and volume should be more
than 14 cm × 14 cm and 250 mL, respectively.
4. Immun-Blot PVDF (Bio-Rad Laboratories, Hercules, CA,
USA): Cut a sheet into 14 cm × 14 cm. Incubate in methanol
for 5 min just before use.
5. Filter paper: 20 cm × 20 cm, for transfer grade.
6. Cy5 Mono reactive dye (GE Healthcare): Dissolve 1 vial with
100
L of dimethylformamide, vortex, and add to 100 mL of
PBS just before use.
7. Wash buffer: 0.05 % Tween-20/PBS. Add 500
μ
μ
L of Tween-20
to 1 L of PBS, mix gently.
8. Blocking buffer: 0.5 % casein/PBS. Dissolve 1 g of casein to
200 mL of PBS. Heat the solution at 60 °C for 1 h, because
casein dissolves with difficulty. After cooling to RT, store
at 4 °C.
9. Horseradish peroxidase (HRP)-linked anti-human IgE (Nordic
Immunology, Tilburg, Netherlands): Dilute 1:1,000 with
0.1 % casein/PBS just before use.
10. ECL Plus (Plex) Western Blotting Detection Reagents (GE
Healthcare): available for detection by a fl uorescence imager.
11. Image scanner: We use Typhoon 9400 from GE Healthcare.
12. Low-fl uorescence glass plate (two plates): Large enough to
cover a 14 cm × 14 cm membrane.
