Biology Reference
In-Depth Information
and place the cassette in a transfer tank. Pour 5 L of transfer
buffer into the tank, and transfer at 180 mA overnight under
cooling (
see
Note 10
).
2. After blotting, the membrane is picked up and incubated with
Cy5/PBS solution to label the transferred proteins with Cy5
(
see
Note 11
). Gently shake for 1 h at RT, avoiding exposure
to light.
3. Wash the membrane with 100 mL of methanol. Gently shake
for 10 min at RT and repeat two times with fresh methanol.
4. Wash the membrane with 100 mL of 0.05 % Tween-20/PBS.
Gently shake for 10 min at RT and repeat two times with fresh
solution.
5. Block the membrane with 100 mL of 0.5 % casein/PBS. Gently
shake for 2 h at RT.
6. Pipette diluted serum (
see
Note 12
) on the transferred side of
the membrane. Incubate the membrane for 1 h at RT and then
overnight at 4 °C under gentle shaking.
7. Wash the membrane with 100 mL of 0.05 % Tween-20/PBS
(
see
Note 13
). Gently shake for 10 min at RT and repeat two
times with fresh solution.
8. Incubate the membrane in HRP-linked anti-human IgE
(1:1,000 diluted with 0.1 % casein/PBS) solution for 1.5 h at
RT under gentle shaking.
9. Wash the membrane with 100 mL of 0.05 % Tween-20/PBS.
Gently shake for 10 min at RT and repeat two times with fresh
solution.
10. Pipette ECL plus (plex) reagent on to the membrane, and after
incubation for an appropriate period of time, move the mem-
brane into ultrapure water (
see
Note 14
).
11. Place the membrane on a clean, low-fl uorescence glass plate
with the stained side facing down. Gently pour a few milliliters
of ultrapure water on the surface of the membrane and cover it
with another clean glass plate (Fig.
1
,
see
Note 15
).
12. Place the gel plate on the scanning table of the image scanner.
Scan gel images of Cy2 (458 nm, band-pass 40) for ECL plus
(plex), and Cy5 (670 nm, band-pass 30) (Fig.
2
).
1. Prepare a gel separating 100
g of proteins, stain with
Coomassie brilliant blue or silver stain.
2. Excise gel pieces containing IgE-binding proteins from the
2D-gel and place them, one each in individual 1.5-mL centri-
fuge tubes.
3. The gel pieces are then decolorized with 50
μ
3.4 In Gel Digestion
of Protein Spots
L of decolorizing
buffer each. Replace fresh buffer and repeat washing the gel
pieces until they are no longer stained.
μ
